Cattle reproductive disease vaccines

ABSTRACT

The present invention relates to combination vaccines and methods for treating or preventing diseases or disorders in an animal caused by infection by Bovine Viral Diarrhea Virus (BVDV) Types 1 and 2, Bovine Herpes Virus Type-1 (BHV-1), Bovine Respiratory Syncytial Virus (BRSV), Parainfluenza Virus (PI 3 ),  Campylobacter fetus, Leptospira canicola, Leptospira grippotyphosa, Leptospira hardj - prajitno, Leptospira icterohaemmorrhagiae, Leptospira hardjo - bovis  and  Leptospira pomona  by administering to the animal an effective amount of a combination vaccine. The combination vaccine can be a whole or partial cell inactivated or modified live preparation.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of copending application Ser.No. 10/647,919 filed on Aug. 26, 2003 which claims benefit of U.S.Provisional Application No. 60/405,969 filed Aug. 26, 2002.

FIELD OF THE INVENTION

The present invention relates to combination vaccines and methods fortreating or preventing diseases or disorders in an animal caused byinfection by Bovine Viral Diarrhea Virus (BVDV) Types 1 and 2, BovineHerpes virus Type-1 (BHV-1), Bovine Respiratory Syncytial Virus (BRSV),Parainfluenza Virus (PI₃), Campylobacter fetus, Leptospira canicola,Leptospira grippotyphosa, Leptospira borgpetersenii hardjo-prajitno,Leptospira icterohaemmorrhagiae, Leptospira borgpetersenii hardjo-bovisand Leptospira interrogans pomona by administering to the animal aneffective amount of a combination vaccine. The combination vaccine canbe a whole or partial cell inactivated or modified live preparation.

BACKGROUND OF THE INVENTION

Five viral agents associated with the bovine respiratory disease (BRD)complex—Bovine Herpes Virus Type-1 (BHV-1), also known as infectiousbovine rhinotracheitis virus (IBR), Bovine viral diarrhea virus (BVDV)Types 1 and 2, Bovine Respiratory Syncytial Virus (BRSV), andParainfluenza Virus (PI₃), cause respiratory and reproductive systeminfections of great economic importance to the cow-calf and dairyindustries worldwide. BRD causes a broad array of clinical syndromesincluding acute onset respiratory disease and abortion. The respiratoryform of BRD is characterized by inflammation, swelling, hemorrhage, andnecrosis of the mucous membranes of the respiratory tract and may beaccompanied by high fever, anorexia, depression, nasal discharge,labored breathing, and inflamed muzzle. Abortions induced by IBR andBVDV virus can occur in all three trimesters, but chiefly during thelast half of gestation, and often without evidence of other clinicalsigns (Ellis et al. (1996) JAVMA 208:393-400; Ellsworth et al. (1994)In: Proceedings, 74^(th) Conference of Research Workers in AnimalDisease: 34).

Bovine Herpes Virus Type-1 (BHV-1), is a member of thealphaherpesviridae subfamily, and produces a variety of clinical formsof disease in cattle, including respiratory and genital infections,conjunctivitis, encephalitis, and abortions. Previous attempts atcontrolling BHV-1 infection have utilized vaccines comprising liveattenuated virus (Gerber, J. D., et al., 1978, Am. J. Vet. Res.39:753-760; Mitchell, D., 1974, Can. Vet. Jour. 15:148-151), inactivatedvirus (Frerichs, G. N., et al., 1982, Vet. Rec. 111:116-122), and viralsubunits such as, e.g., one of the three major BHV-1 glycoproteins,which have been designated in the art as gI, gIII, and gIV (Babiuk, L.A., et al., 1987, Virology 159:57-66; van Drunen, S., et al., 1993,Vaccine 11:25-35). In addition, the ability of a recombinant, truncatedversion of the BHV-1 gIV glycoprotein (designated in the art as BHV-1tgIV) to induce mucosal immunity against BHV-1 has been demonstrated(van Drunen, S., et al., 1994, Vaccine, 12:1295-1302). However, theart-recognized BHV-1 vaccines are contraindicated for use in pregnantcattle, seropositive or seronegative, and also contraindicated for usein calves nursing pregnant cows.

BVDV Types 1 and 2 have been implicated in a variety of clinicalsyndromes. Studies have established that the virus causes severe primaryrespiratory disease; that persistently infected (PI) cattle are a majorsource of infection for susceptible calves; and that BVDV infects whitecell reservoirs, causing profound and broad-based deficits in the immunesystem. Ellis et al. (1996) JAVMA 208:393-400; Baum et al. (1993) TheCompendium Collection: Infectious Disease in Food Animal Practice.Trenton, N.J. Veterinary Learning Systems-113-121; Meyling et al. (1987)Agric Pestivirus Infect Rumin 225-231. Abortion or mummification canresult when pregnant cattle become infected especially during the firsttrimester. Bolin et al. (1989) Am J. Vet Res 52:1033-1037. Mucosaldisease, another often fatal manifestation of bovine viral diarrhea(BVD), results from early fetal infection with a noncytopathic BVDVbiotype, development of immunotolerance to the virus, birth of apersistently infected (PI) calf, and subsequent superinfection with acytopathic BVDV biotype. Bolin et al. (1989) Am J. Vet Res 52:1033-1037.BVDV Type 2, once recognized chiefly as a hemorrhagic BVDV isolatemostly in dairy herds, has become the predominant strain isolated inmost regions of the United States from both BVD-related abortions andrespiratory cases. Van Oirschot et al. (1999) Vet Micro 64:169-183.

BVDV is classified in the pestivirus genus and Flaviviridae family. Itis closely related to viruses causing border disease in sheep andclassical swine fever. Infected cattle exhibit “mucosal disease” whichis characterized by elevated temperature, diarrhea, coughing andulcerations of the alimentary mucosa (Olafson, et al., Cornell Vet.36:205-213 (1946); Ramsey, et al., North Am. Vet. 34:629-633 (1953)).The BVD virus is capable of crossing the placenta of pregnant cattle andmay result in the birth of PI calves (Malmquist, J. Am. Vet. Med. Assoc.152:763-768 (1968); Ross, et al., J. Am. Vet. Med. Assoc. 188:618-619(1986)). These calves are immunotolerant to the virus and persistentlyviremic for the rest of their lives. They provide a source for outbreaksof mucosal disease (Liess, et al., Dtsch. Tieraerztl. Wschr. 81:481-487(1974) and are highly predisposed to infection with microorganismscausing diseases such as pneumonia or enteric disease (Barber, et al.,Vet. Rec. 117:459-464 (1985).

According to BVDV virus growth studies in cultured cells, two viralbiotypes have been classified: viruses that induce a cytopathic effect(cp) and viruses that do not induce a cytopathic effect (ncp) ininfected cells (Lee et al., Am. J. Vet. Res. 18: 952-953; Gillespie etal., Cornell Vet. 50: 73-79, 1960). Cp variants can arise from the PIanimals preinfected with ncp viruses (Howard et al., Vet. Microbiol. 13:361-369, 1987; Corapi et al., J. Virol. 62: 2823-2827, 1988). Based onthe genetic diversity of the 5′ non-translated-region (NTR) and theantigenic differences in the virion surface glycoprotein E2 of BVDviruses, two major genotypes have been proposed: type I and II. BVDVtype 1 represents classical or traditional virus strains which usuallyproduce only mild diarrhea in immunocompetent animals, whereas BVDV type2 are emerging viruses with high virulence which can producethrombocytopenia, hemorrhages and acute fatal disease (Corapi et al., J.Virol. 63: 3934-3943; Bolin et al., Am. J. Vet. Res. 53: 2157-2163;Pellerin et al., Virology 203: 260-268, 1994; Ridpath et al., Virology205: 66-74, 1994; Carman et al., J. Vet. Diagn. Invest. 10: 27-35,1998). Type I and II BVDV viruses have distinct antigenicity determinedby a panel of monoclonal antibodies (Mabs) and by cross-neutralizationusing virus-specific antisera raised in animals (Corapi et al., Am. J.Vet. Res. 51: 1388-1394, 1990). Viruses of either genotype may exist asone of the two biotypes, cp or ncp virus.

Studies from BVD virus infected animals suggest that BVD viruses induceboth B-cell and T-cell responses in animals (Donis et al., Virology 158:168-173, 1987; Larsson et al., Vet. Microbiol. 31: 317-325, 1992; Howardet al., Vet. Immunol. Immunopathol. 32: 303-314, 1992; Lambot et al., J.Gen. Virol. 78: 1041-1047, 1997; Beer et al., Vet. Microbiology. 58:9-22, 1997).

A number of BVDV vaccines have been developed using chemicallyinactivated BVD viral isolates (Fernelius et al., Am. J. Vet. Res. 33:1421-1431, 1972; Kolar et al., Am. J. Vet. Res. 33: 1415-1420, 1972;McClurkin et al., Arch. Virol. 58: 119, 1978). Multiple doses arerequired for the inactivated viral vaccines to achieve primaryimmunization. Some inactivated BVDV vaccines provide protection againstinfection by type I BVDV only (Beer et al., Vet. Microbiology.77:195-208, 2000). Fetal protection has not been achieved withinactivated BVDV vaccines due to a short duration of immunity and aninefficient cross-type protection (Bolin, Vet. Clin. North Am. FoodAnim. Pract. 11: 615-625, 1995).

Modified-live virus (MLV) vaccines, on the other hand, offer a higherlevel of protection. Currently, licensed BVDV MLV vaccines are producedusing attenuated viruses obtained via repeated passage in bovine orporcine cells (Coggins et al., Cornell Vet. 51: 539-, 1961; Phillips etal., Am. J. Vet. Res. 36:135-, 1975), or using chemically modifiedviruses which exhibit a temperature-sensitive phenotype (Lobmann et al.,Am. J. Vet. Res. 45: 2498-, 1984; 47: 557-561, 1986). A single dose ofMLV vaccine is sufficient for immunization, and duration of the immunitycan last for years in vaccinated cattle. However, as these vaccines havebeen developed using type I BVDV virus strains, the protection isagainst type I virus only. Moreover, the available BVDV vaccines are notindicated for use in pregnant cattle or calves nursing pregnant cows.

PI₃ virus typically produces only mild disease when acting alone;however, the virus predisposes the respiratory tract to secondaryinfection with more pathogenic organisms including IBR virus, BRSV, andBVDV, resulting in the classic shipping fever syndrome. Of the variousviruses known to cause respiratory disease in cattle, PI₃ virus is themost widespread. Ellis et al. (1996) JAVMA 208:393-400.

BRSV has a preference for the lower respiratory tract, and severity ofinfection is determined chiefly by the immune system's response to keyviral proteins. Bolin et al. (1990) Am J Vet Res 51:703. Affected cattlegenerally show nonspecific signs including serous nasal and oculardischarge, a mild, often biphasic fever, and dry, hacking cough. Moreseverely affected cattle develop a harsh cough, show labored, open-mouthbreathing, and frothy saliva around the mouth, and may quit eating anddrinking. Ellis et al. (1996) JAVMA 208:393-400.

Leptospirosis, caused by spirochetes of the genus Leptospira, is aneconomically important zoonotic infection of livestock. Leptospiraborgpetersenii serovar hardjo (L. hardjo) and L. interrogans serovarpomona (L. pomona) are the two serovars most commonly associated withcattle leptosporosis worldwide. In one survey of US cattle, 29% reactedserologically with L. hardjo, and 23% with L. pomona. Leptospires invadethe body via mucous membranes or broken skin, and are disseminated viathe blood. They display tropisms for the kidney and genital tract, andless commonly the vitreous humor of the eye and the central nervoussystem. The most common means of infection is by direct or indirectcontact with infected urine, milk, or placental fluids, but venereal andtrans-ovarian transmission are also known. Leptospiral infection ofcattle may result in acute fever, agalactia, abortion, or birth ofpremature and weak infected calves, and may contribute to breedingfailures and low conception rates. Infections can be treated withantibiotics, but they may be in apparent in cattle that are notlactating or pregnant. In such cattle they establish acute or chronicinfection of the kidneys, resulting in urinary shedding of virulentorganisms which in turn may infect other animals or their humanhandlers. Immunity to Leptospira is serovar specific, and althoughvaccines have been available for many years, most induce only a poor andshort-lived immunity.

There is therefore a need for development of combination vaccines thatprovide protection against a large variety of antigens that are safe forpregnant and nursing cows and their offspring and meet dairy and beefcow market needs. The present invention provides vaccines for thetreatment and prevention of the major infectious causes of respiratoryand reproductive disease in animals, such as cows and calves. Thepresent invention further provides immunogenic compositions and methodsof treating or preventing diseases or disorders in animals.

SUMMARY OF THE INVENTION

The present invention provides a method of treating or preventing adisease or disorder in an animal caused by infection with at least oneof, BVDV Type 1 or Type 2, BHV-1, PI3, BRSV, Campylobacter fetus,Leptospira canicola, Leptospira grippotyphosa, Leptospira borgpeterseniihardjo-prajitno, Leptospira icterohaemmorrhagiae, Leptospiraborgpetersenii hardjo-bovis and Leptospira interrogans pomona comprisingadministering to the animal, an effective amount of a combinationvaccine.

The present method provides protection to animals such as bovine, inparticular, dairy cattle, against respiratory infection and reproductivedisease. The present method provides protection to animals such aspregnant cows against abortion caused by IBR and persistent fetalinfections caused by BVDV, Types 1 and 2. The present method alsoprovides protection to animals such as lactating cows and calves nursingpregnant cows against persistent infections caused by BVDV, Types 1 and2. Thus, the present method provides protection to breeding age animals,pregnant animals and lactating animals.

The combination vaccine employed in the present methods can be a wholeor partial cell preparation (e.g., modified live preparation). Thecombination vaccine administered in accordance with the presentinvention may include additional components, such as an adjuvant andoptionally a second or more antigens. A second antigen is selected fromthe following, including, but not limited, to bovine herpesvirus type 1(BHV-1), bovine viral diarrhea virus (BVDV type 1 or 2), bovinerespiratory syncitial virus (BRSV), parainfluenza virus (PI3),Leptospira canicola, Leptospira grippotyphosa, Leptospira borgpeterseniihardio-prajitno, Leptospira icterohaemmorrhagia, Leptospira interroganspomona, Leptospira borgpetersenii hardjo-bovis, Leptospira bratislava,Campylobacter fetus, Neospora caninum, Trichomonus fetus, Mycoplasmabovis, Haemophilus somnus, Mannheimia haemolytica and Pasturellamultocida.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of treating or preventing adisease or disorder in an animal caused by infection with IBR, BVDV,PI3, BRSV, Campylobacter fetus and/or Leptospirae by administering tothe animal, an effective amount of a combination vaccine.

In certain embodiments, the vaccines used in the method of the presentinvention comprise a modified live vaccine and a pharmaceuticallyacceptable carrier, or a modified live vaccine and an adjuvant.

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the following subsectionswhich describe or illustrate certain features, embodiments orapplications of the invention.

Definitions and Abbreviations

The term “treating or preventing” with respect to a disease or disorderas used herein means reducing or eliminating the risk of infection by avirulent BVDV virus, types I and 2; IBR; PI3; BRSV; Campylobacteria;and/or Leptospira antigens, ameliorating or alleviating the symptoms ofan infection, or accelerating the recovery from an infection. Thetreatment is considered therapeutic if there is a reduction in viral orbacterial load, decrease in pulmonary infections, reduced rectaltemperatures, and/or increase in food uptake and/or growth. Thetreatment is also considered therapeutic if there is a reduction infetal infection and urinary shedding due to infection with Leptospiraserovars hardjo and pomona, for example.

The method of the present invention is, for example, effective inpreventing or reducing abortion caused by IBR and infections caused byBVDV Types 1 and 2, and reducing rectal temperatures. The presentinvention is therefore contemplated to provide fetal protection againstIBR and infections caused by BVDV Types 1 and 2 as well as fetalprotection against cattle herpes and cattle pestiviruses. The presentinvention is also contemplated to provide protection against persistentfetal infection, such as persistent BVDV infection. By “persistent fetalinfection” is meant infection occurring during early fetal development(e.g., 45-125 days of gestation) that leads to the live birth of animalsthat are immunotolerant to BVDV and maintain active BVDV replication andmultiplication that often occurs at a high rate for months or years,serving as a permanent source of BVDV in the herd. These persistentlyinfected animals are also at risk of developing fatal mucosal disease ifsuperinfected with a cytopathic virus biotype.

The term “combination vaccine” is meant a bivalent or multivalentcombination of antigens including modified live antigens and/orinactivated antigens. In accordance with the present invention acombination vaccine can comprise modified live infectious IBR, PI3, BRSVand inactivated BVDV Types 1 and 2, one or more antigens such as but notlimited to Leptospira canicola, Leptospira grippotyphosa, Leptospiraborgpetersenii hardio-prajitno, Leptospira icterohaemmorrhagia,Leptospira interrogans pomona, Leptospira borgpetersenii hardjo-bovis,Leptospira bratislava, Campylobacter fetus, Neospora caninum,Trichomonus fetus, Mycoplasma bovis, Haemophilus somnus, Mannheimiahaemolytica and Pasturella multocida, a veterinary acceptable carrierand an adjuvant. In a preferred embodiment the modified live IBRcomponent is temperature sensitive IBR. In another preferred embodimentthe BVDV Type 2 component is cytopathic (cpBVD-2 strain 53637-ATCC No.PTA-4859) and the BVDV Type 1 component is cytopathic 5960 (cpBDV-1strain 5960—National Animal Disease Center, United States Department ofAgriculture, Ames, Iowa). The present invention also contemplatesnon-cytopathic BVDV Type 1 and Type 2 strains. In still anotherpreferred embodiment, the modified live antigens are desiccated,lyophilized or vitrified.

In accordance with the present invention a combination vaccine cancomprise inactivated BVDV Types 1 and 2, one or more antigens such as,but not limited to, Leptospira canicola, Leptospira grippotyphosa,Leptospira borgpetersenii hardio-prajitno, Leptospiraicterohaemmorrhagia, Leptospira interrogans pomona, Leptospiraborgpetersenii hardjo-bovis, Leptospira bratislava, Campylobacter fetus,Neospora caninum, Trichomonus fetus, Mycoplasma bovis, Haemophilussomnus, Mannheimia haemolytica and Pasturella multocida, a veterinaryacceptable carrier and an adjuvant. The term “combination vaccine” asused herein also refers to a multicomponent composition containing atleast one modified live antigen, at least one second antigen and anadjuvant which prevents or reduces the risk of infection and/or whichameliorates the symptoms of infection. In a preferred embodiment thesecond antigen is inactivated. In a preferred embodiment the source ofthe combination vaccine is PregSure® 5 (Pfizer, Inc.), PregSure® 5-L5(Pfizer, Inc.) and PregSure® 5-VL5 (Pfizer, Inc.). A particularlypreferred source of the combination vaccine is PregSure® 5-VL5.

The protective effects of a combination vaccine composition against apathogen are normally achieved by inducing in the subject an immuneresponse, either a cell-mediated or a humoral immune response or acombination of both. Generally speaking, abolished or reduced incidencesof BVDV, IBR, and/or PI3 infection, amelioration of the symptoms, oraccelerated elimination of the viruses from the infected subjects areindicative of the protective effects of a combination vaccinecomposition. The vaccine compositions of the present invention provideprotection against infections caused by either or both type 1 and type 2BVD viruses as well as abortions caused by BHV-1 (IBR) and respiratoryinfections caused by PI3 and BRSV.

The present method of treating or preventing a disease or disorder in ananimal caused by infection with IBR, BVDV, PI3, BRSV, Campylobacterfetus and/or Leptospirae by administering a combination vaccine is alsoreferred to herein as a vaccination method.

The term “combination vaccine” that may be used in the present methodcan include, for example, an inactivated whole or partial C. fetusand/or Leptospira cell preparation, inactivated BVDV types 1 and 2and/or one or more modified live antigens such as BHV-1, PI3 and/orBRSV.

In one embodiment, the vaccine compositions of the present inventioninclude an effective amount of one or more of the above-described BVDVviruses, preferably cpBVD-2 strain 53637 (ATCC No. PTA-4859); cpBVD-1strain 5960 (cpBDV-1 strain 5960—National Animal Disease Center, UnitedStates Department of Agriculture, Ames, Iowa); IBR ts mutant strain RBL106 (National Institute of Veterinary Research, Brussels, Belgium); PI₃ts mutant strain RBL 103 (RIT, Rixensart, Belgium); BRSV strain 375(Veterinary Medical Research Institute, Ames, Iowa) Purified BVDVviruses can be used directly in a vaccine composition, or preferably,BVD viruses can be further attenuated by way of chemical inactivation orserial passages in vitro. Typically, a vaccine contains between about1×10³ and about 1×10¹⁰ plaque or colony forming units of virus, with aveterinary acceptable carrier and an adjuvant, in a volume of between0.5 and 5 ml and preferably about 2 ml. The precise amount of a virus ina vaccine composition effective to provide a protective effect can bedetermined by a skilled veterinary physician. Veterinary acceptablecarriers suitable for use in vaccine compositions can be any of thosedescribed hereinbelow.

The typical route of administration will be intramuscular orsubcutaneous injection of between about 0.1 and about 5 ml of vaccine.The vaccine compositions of the present invention can also includeadditional active ingredients such as other vaccine compositions againstBVDV, e.g., those described in WO 9512682, WO 9955366, U.S. Pat. No.6,060,457, U.S. Pat. No. 6,015,795, U.S. Pat. No. 6,001,613, and U.S.Pat. No. 5,593,873.

Vaccination can be accomplished by a single inoculation or throughmultiple inoculations. If desired, sera can be collected from theinoculated animals and tested for the presence of antibodies to BVDvirus.

In another embodiment of the present invention, the vaccine compositionsare used in treating BVDV infections. Accordingly, the present inventionprovides methods of treating infections in animal subjects caused by BVDviruses of type 1 or type 2, or a combination of type 1 and type 2, byadministering to an animal, a therapeutically effective amount of a BVDvirus of the present invention. In another embodiment the vaccinecompositions of the present invention are effective for the improvementof herd fertility, and for the reduction of the risk of diseasetransmission from cattle to human handlers.

By “animal subject” is meant to include any animal that is susceptibleto BVDV, BHV, PI₃, BRSV or Leptospira infections, for example, such asbovine, sheep and swine.

In practicing the present methods, a vaccine composition of the presentinvention is administered to a cattle preferably via intramuscular orsubcutaneous routes, although other routes of administration can be usedas well, such as e.g., by oral, intranasal (e.g. aerosol or otherneedleless administration), intra-lymph node, intradermal,intraperitoneal, rectal or vaginal administration, or by a combinationof routes. Boosting regimens may be required and the dosage regimen canbe adjusted to provide optimal immunization.

By “immunogenic” is meant the capacity of a BVD virus to provoke animmune response in an animal against type 1 or type 2 BVD viruses, oragainst both type 1 and type 2 BVD viruses. The immune response can be acellular immune response mediated primarily by cytotoxic T-cells, or ahumoral immune response mediated primarily by helper T-cells, which inturn activates B-cells leading to antibody production.

According to the present invention, the viruses are preferablyattenuated by chemical inactivation or by serial passages in cellculture prior to use in an immunogenic composition. The methods ofattenuation are well known to those skilled in the art.

A preferred virus to be included in an immunogenic composition of thepresent invention is BVDV cp53637(ATCC No. PTA-4859). Another preferredvirus to be included in an immunogenic composition of the presentinvention is BVDV 5960. A further preferred virus to be included in animmunogenic composition of the present invention is IBR strain ts mutantstrain RBL 106. Another preferred virus to be included in an immunogeniccomposition of the present inventions is PI₃ ts mutant strain RBL 103.Yet another preferred virus to be included in an immunogenic compositionof the present invention is BRSV strain 375.

The immunogenic compositions of the present invention can also includeadditional active ingredients such as other immunogenic compositionsagainst BVDV, e.g., those described in copending application Ser. No.08/107,908, WO 9512682, WO 9955366, U.S. Pat. No. 6,060,457, U.S. Pat.No. 6,015,795, U.S. Pat. No. 6,001,613, and U.S. Pat. No. 5,593,873.

In addition, the immunogenic and vaccine compositions of the presentinvention can include one or more veterinary-acceptable carriers. Asused herein, “a veterinary-acceptable carrier” includes any and allsolvents, dispersion media, coatings, adjuvants, stabilizing agents,diluents, preservatives, antibacterial and antifungal agents, isotonicagents, adsorption delaying agents, and the like. Diluents can includewater, saline, dextrose, ethanol, glycerol, and the like. Isotonicagents can include sodium chloride, dextrose, mannitol, sorbitol, andlactose, among others. Stabilizers include albumin, among others.Adjuvants include, but are not limited to, the RIBI adjuvant system(Ribi Inc.), alum, aluminum hydroxide gel, Cholesterol, oil-in wateremulsions, water-in-oil emulsions such as, e.g., Freund's complete andincomplete adjuvants, Block co-polymer (CytRx, Atlanta Ga.), SAF-M(Chiron, Emeryville Calif.), AMPHIGEN® adjuvant, saponin, Quil A, QS-21(Cambridge Biotech Inc., Cambridge Mass.), GPI-0100 (GalenicaPharmaceuticals, Inc., Birmingham, Ala.) or other saponin fractions,monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat-labileenterotoxin from E. coli (recombinant or otherwise), cholera toxin, ormuramyl dipeptide, among many others. The immunogenic compositions canfurther include one or more other immunomodulatory agents such as, e.g.,interleukins, interferons, or other cytokines. The immunogeniccompositions can also include Gentamicin and Merthiolate. While theamounts and concentrations of adjuvants and additives useful in thecontext of the present invention can readily be determined by theskilled artisan, the present invention contemplates compositionscomprising from about 50 μg to about 2000 μg of adjuvant and preferablyabout 500 μg/2 ml dose of the vaccine composition. In another preferredembodiment, the present invention contemplates vaccine compositionscomprising from about 1 μg/ml to about 60 μg/ml of antibiotic, and morepreferably less than about 30 μg/ml of antibiotic.

The immunogenic compositions of the present invention can be made invarious forms depending upon the route of administration. For example,the immunogenic compositions can be made in the form of sterile aqueoussolutions or dispersions suitable for injectable use, or made inlyophilized forms using freeze-drying techniques. Lyophilizedimmunogenic compositions are typically maintained at about 4° C., andcan be reconstituted in a stabilizing solution, e.g., saline or andHEPES, with or without adjuvant.

The immunogenic compositions of the present invention can beadministered to animal subjects to induce an immune response againsttype 1 or type 2 BVD viruses, or against both type 1 and type 2 BVDviruses. Accordingly, another embodiment of the present inventionprovides methods of stimulating an immune response against type 2 ortype 2 BVD viruses, or against a combination of type 1 and type 2 BVDviruses by administering to an animal subject an effective amount of animmunogenic composition of the present invention described above. By“animal subject” is meant to include any animal that is susceptible toBVDV infections, such as bovine, sheep and swine.

In accordance with the methods of the present invention, a preferredimmunogenic composition for administration to an animal subject includesthe BVDV cp53637 virus and/or the BVDV cp5960 virus. An immunogeniccomposition containing a BVDV virus, preferably attenuated by chemicalinactivation or serial passage in culture, is administered to a cattlepreferably via intramuscular or subcutaneous routes, although otherroutes of administration can be used as well, such as e.g., by oral,intranasal, intra-lymph node, intradermal, intraperitoneal, rectal orvaginal administration, or by a combination of routes.

Immunization protocols can be optimized using procedures well known inthe art. A single dose can be administered to animals, or,alternatively, two or more inoculations can take place with intervals oftwo to ten weeks. Depending on the age of the animal, the immunogenic orvaccine composition can be readministered. For example, the presentinvention contemplates the vaccination of healthy cattle prior to sixmonths of age and revaccination at six months of age. In anotherexample, the present invention contemplates the vaccination ofprebreeding cattle at about 5 weeks prebreeding and again at about 2weeks prebreeding to protect a fetus against infection caused by BVDVTypes 1 and 2. Semiannual revaccination with a single dose of thecombination vaccine is also contemplated to prevent BVDV fetalinfection.

The extent and nature of the immune responses induced in the cattle canbe assessed by using a variety of techniques. For example, sera can becollected from the inoculated animals and tested for the presence ofantibodies specific for BVDV viruses, e.g., in a conventional virusneutralization assay.

The term “cattle” as used herein refers to bovine animals including butnot limited to steer, bulls, cows, and calves. Cattle as used hereinrefers to pregnant and lactating bovine animals. Preferably, the methodof the present invention is applied to an animal which is a non-humanmammal; preferably, a lactating or pregnant cow and its fetus.

The term “therapeutically effective amount” or “effective amount” refersto an amount of combination vaccine sufficient to elicit an immuneresponse in the animal to which it is administered. The immune responsemay comprise, without limitation, induction of cellular and/or humoralimmunity. The amount of a vaccine that is therapeutically effective mayvary depending on the particular virus used, the condition of the cattleand/or the degree of infection, and can be determined by a veterinaryphysician.

Inactivated (Partial or Whole Cell) and Modified Live Vaccines

Inactivated or modified live vaccines for use in the method of thepresent invention can be prepared using a variety of methods which areknown in the art.

For example, BVDV isolates can be obtained directly from infected cowuteri using known techniques.

BVDV isolates can be inactivated using a variety of known methods, e.g.,treating the bacterial isolate with binary ethyleneimine (BEI) asdescribed in U.S. Pat. No. 5,565,205, or inactivation with formalin,glutaraldehyde, heat, irradiation, BPL, or other inactivating agentsknown to the art.

In addition to inactivated viral isolates, a vaccine product can alsoinclude an appropriate amount of one or more commonly used adjuvants.Suitable adjuvants may include, but are not limited to: mineral gels,e.g., aluminum hydroxide; surface active substances such aslysolecithin; glycosides, e.g., saponin derivatives such as Quil A orGPI-0100; pluronic polyols; polyanions; non-ionic block polymers, e.g.,Pluronic F-127 (B.A.S.F., USA); peptides; mineral oils, e.g. MontanideISA-50 (Seppic, Paris, France), carbopol, Amphigen, Amphigen Mark II(Hydronics, USA), Alhydrogel, oil emulsions, e.g. an emulsion of mineraloil such as BayolF/Arlacel A and water, or an emulsion of vegetable oil,water and an emulsifier such as lecithin; alum; bovine cytokines;cholesterol; and combinations of adjuvants. In a preferred embodiment,the saponin containing oil-in-water emulsion is conventionallymicrofluidized.

A particularly preferred source of BVDV type 1, for use in the vaccineand method of the present invention is PregSure® (PFIZER INC.),containing BVDV strain 5960 (acquired from the National Animal DiseaseCenter (NADC), USDA, Ames, Iowa). A particularly preferred source ofBVDV type 2, for use in the vaccine and method of the present inventionis PregSure® (Pfizer, Inc.), containing BVDV strain 53637 (ATCC No.PTA-4859), acquired from the University of Guelph, Guelph, Ontario.

Preferably, the strains 5960 and 53637 are inactivated with BEI andadjuvanted with a commercially available adjuvant, preferably, QuilA-Cholesterol-Amphigen (Hydronics, USA). A preferred dose of theimmunogenic and vaccine compositions of the present invention is about2.0 ml. Preservatives can be included in the methods and compositions ofthe present invention. Preservatives contemplated by the presentinvention include gentamicin and merthiolate. A carrier can also beadded, preferably, PBS. Preparation of modified live vaccines, such asby attenuation of virulent strains by passage in culture, is known inthe art.

Inactivated BVDV isolates can also be combined with the followingbacteria and viruses, including but not limited to, bovine herpesvirustype 1 (BHV-1), bovine respiratory syncitial virus (BRSV), parainfluenzavirus (PI3), Campylobacter fetus, Leptospira canicola, Leptospiragrippotyphosa, Leptospira borgpetersenii hardjo-prajitno, Leptospiraicterohaemmorrhagiae, Leptospira borgpetersenii hardjo-bovis andLeptospira interrogans pomona.

Dosing and Modes of Administration

According to the present invention, an effective amount of a combinationvaccine administered to cattle, including pregnant cows and calvesnursing pregnant cows provides effective immunity against disease andfetal infection associated with Bovine Viral Diarrhea Virus (Type 1 and2). In one embodiment, the combination vaccine is administered to calvesin two doses at an interval of about 3 to 4 weeks. For example, thefirst administration is performed when the animal is about 1 to about 3months of age. The second administration is performed about 1 to about 4weeks after the first administration of the combination vaccine.

In a preferred embodiment, the first administration is performed about 5weeks prior to animal breeding. The second administration is performedabout 2 weeks prior to animal breeding. Administration of subsequentvaccine doses is preferably done on an annual basis. In anotherpreferred embodiment, animals vaccinated before the age of about 6months should be revaccinated after 6 months of age. Administration ofsubsequent vaccine doses is preferably done on an annual basis.

The amount of combination vaccine that is effective depends on theingredients of the vaccine and the schedule of administration.Typically, when an inactivated Bovine Viral Diarrhea Virus preparationis used in a vaccine, an amount of the vaccine containing about 10³ toabout 10¹⁰ colony forming units per dose of BVDV, and preferably about10⁵ to about 10⁸ colony forming units per dose of BVDV (Type 1 and 2) iseffective when administered twice to the animal during a period of about3 to 4 weeks. Preferably, a combination vaccine that provides effectiveimmunity contains about 10⁵ to 10⁸ colony forming units/dose of BVDV(Type 1 and 2) and more preferably, about 10⁶ colony forming units/dose,when administered twice to the animal during a period of about 3 to 4weeks. The first administration is performed about 5 weeks prior toanimal breeding. The second administration is performed about 2 weeksprior to animal breeding. Administration of subsequent vaccine doses ispreferably done on an annual basis. Animals vaccinated before the age ofabout 6 months should be revaccinated after 6 months of age.Administration of subsequent vaccine doses is preferably done on anannual basis.

According to the present invention, when the preferred product,PregSure® 5 (Pfizer, Inc.), is administered, PregSure® 5 is administeredpreferably twice, each time in the amount of about 0.5 ml to about 5.0ml, preferably about 1.5 ml to about 2.5 ml, and more preferably, about2 ml. When the preferred product PregSure® 5-L5 or PregSure® 5-VL5 isadministered, PregSure® 5-L5 or PregSure® 5-VL5 is administeredpreferably twice, each time in the amount of about 0.5 ml to about 10.0ml, preferably about 3 ml to about 7 ml, and more preferably, about 5ml. The first administration is performed about 5 weeks prior to animalbreeding. The second administration is performed about 2 weeks prior toanimal breeding. Administration of subsequent vaccine doses ispreferably done on an annual basis. Animals vaccinated before the age ofabout 6 months should be revaccinated after 6 months of age.Administration of subsequent vaccine doses is preferably done on anannual basis.

In accordance with the present invention, administration can be achievedby known routes, including the oral, intranasal, topical, transdermal,and parenteral (e.g., intravenous, intraperitoneal, intradermal,subcutaneous or intramuscular). A preferred route of administration issubcutaneous or intramuscular administration.

The present invention also contemplates a single primary dose followedby annual revaccination, which eliminates the necessity ofadministration of additional doses to calves prior to annualrevaccination in order to generate and/or maintain immunity againstinfection.

The combination vaccine administered in accordance with the presentinvention can include additional components, such as an adjuvant (e.g.,mineral gels, e.g., aluminum hydroxide; surface active substances suchas Cholesterol, lysolecithin; glycosides, e.g., saponin derivatives suchas Quil A, QS-21 or GPI-0100; pluronic polyols; polyanions; non-ionicblock polymers, e.g., Pluronic F-127; peptides; mineral oils, e.g.Montanide ISA-50, carbopol, Amphigen, Alhydrogel, oil emulsions, e.g. anemulsion of mineral oil such as BayolF/Arlacel A and water, or anemulsion of vegetable oil, water and an emulsifier such as lecithin;alum; bovine cytokines; and combinations of adjuvants.).

According to the present invention, the administration of an effectiveamount of a combination vaccine administered to cattle at approximately3 months of age provides effective immunity against respiratoryinfections and reproductive disease, and reduces abortions. The presentinvention also provides a method of immunizing cattle, including but notlimited to cows, calves, and prebreeding heifers, against infectioncaused by BVDV (types 1 and 2), and respiratory disease attributed toIBR, BVDV (Types 1 and 2), PI3, BRSV, campylobacteriosis andleptospiriosis comprising administering to the animal at least one dose,and preferably two doses of the combination vaccine in order to immunizethe animal against infection caused by BVD (types 1 and 2), IBR, PI3,BRSV, Leptospira canicola, Leptospira grippotyphosa, Leptospiraborgpetersenii hardio-prajitno, Leptospira icterohaemmorrhagia,Leptospira interrogans pomona, Leptospira borgpetersenii hardjo-bovis,Leptospira bratislava, and Campylobacter fetus.

In a preferred embodiment, the vaccine is administered subcutaneously.In another preferred embodiment, the vaccine is administeredintramuscularly. Moreover, it is preferred that the vaccine dosecomprise about 2 ml to about 7 ml, and preferably about 5 ml, each mlcontaining about 10³ to about 10¹⁰ colony forming units/per dose ofvirus. In another preferred embodiment the vaccine comprises about 2 ml,each ml containing about 10³ to about 10¹⁰ colony forming units per doseof virus. The combination vaccine is desirably administered twice to theanimal; once at about 1 to about 3 months of age, and once at about 1 to4 weeks later. The present invention also contemplates semiannualrevaccinations with a single dose and revaccination prior to breeding.

The present invention also provides a method of protecting bovinefetuses against fetal infection and persistent fetal infection,comprising administering to the animal at least one dose, and preferablytwo doses of the combination vaccine in order to immunize the fetusagainst infection caused by BVD (types 1 and 2), IBR, PI3, BRSV,Leptospira canicola, Leptospira grippotyphosa, Leptospira borgpeterseniihardio-prajitno, Leptospira icterohaemmorrhagia, Leptospira interroganspomona, Leptospira borgpetersenii hardjo-bovis, Leptospira bratislava,and Campylobacter fetus. The combination vaccine is desirablyadministered twice to the animal, once about five weeks prior tobreeding and once at about two weeks prior to breeding.

The present invention also contemplates that the administration of aneffective amount of a combination vaccine administered to animals, andpreferably cattle to treat or prevent disorders including persistentfetal infections and reproductive disorders, such as abortions in suchanimals.

The present invention is further illustrated, but not limited by thefollowing examples.

EXAMPLE 1 Materials and Methods

Animals—Fifty-six BVDV seronegative (i.e., having serum neutralization[SN] titers <1:2) cows suitable for breeding were obtained from multiplesources and maintained in research isolation facilities for the durationof the study. Each animal was identified with duplicate ear tags, oneplaced in each ear. New tags were installed in cases where an animallost an ear tag. Prior to the study, test animals were inoculated withcommercial vaccines for leptospirosis, campylobacteriosis (vibriosis),and clostridial infections. Test animals were maintained undersupervision of an attending veterinarian, who clinically monitored themon a daily basis.

Test Vaccine—

The test vaccine was a multivalent, modified live infectious bovinerhinotracheitis (IBR)-parainfluenza 3 (PI3)-respiratory syncytial virus(RSV) vaccine in desiccated form, rehydrated with an inactivated, liquidBVDV vaccine combined with an adjuvant. (Pfizer Inc, New York, N.Y.) TheBVDV component contained the minimum BVDV-1 and -2 immunizing doses,combined with a sterile adjuvant. Potency of the BVDV immunizingantigens was established by calculating the geometric mean titer (GMT)for 8 replicate titrations of the bulk fluid used for vaccinepreparation. Following rehydration, the IBR-PI3-BRSV-BVDV vaccine wasadministered in 2 mL doses by either intramuscular (IM) or subcutaneous(SC) injection. The desiccated IBR-PI3-RSV vaccine reconstituted withsterile water was used as a placebo, and was given by IM injection.

Challenge Virus—A noncytopathic BVDV type 2 field isolate (Strain94B-5359, obtained from Dr. Hana Van Campen, Wyoming State VeterinaryLaboratory, University of Wyoming) was used as a challenge agent. Virusidentity was confirmed by SN assay and reverse transcriptase polymerasechain reaction (RT-PCR). The RT-PCR analysis was positive for BVDV type2 nucleotide sequences for the p125 protein and the 5 untranslatedregion, and negative for the BVDV type 1 gp53 and p80 conservedsequences. Challenge virus potency was established at a GMT of 10^(3.2)TCID₅₀/mL by 2 replicate titrations made immediately before and afterchallenge. Challenge inoculum was given intranasally in a 4 mL divideddose, 2 mL per nostril.

Serologic Assays—Serum neutralization titers for BVDV types 1 and 2 weredetermined by a constant-virus, decreasing-serum assay in bovine cellculture. Serial dilutions of serum were combined with either 50-300TCID₅₀ of cytopathic BVDV type 1 strain 5960, or a similar amount ofcytopathic BVDV type 2 strain 125c.

Virus Isolation—Postchallenge (PC) isolation of BVDV in bovine cellculture was attempted from peripheral cow blood, amniotic fluid, fetalblood, and fetal tissues. A BVDV-positive cell culture was determined byindirect immunofluorescence using goat anti-BVDV polyclonal antibodies.Isolation of BVDV from fetal tissues was also attempted usingimmunohistochemistry methods previously described. (Haines D M, Clark EG, and Dubovi E J. Vet Pathol 1992; 29:27-32.) Whole blood from cows wasdrawn from the jugular vein in 5-10 mL samples and placed inheparin-containing tubes for preparation of buffy coat cells used forvirus isolation attempts. Amniotic fluid was collected under localanesthesia by left-flank laparotomy and aspiration of a 3-5 mL samplefrom the uterus.

Following caesarian section or spontaneous abortion, the eyes, spleen,thymus, and 3 brain sections (brainstem/midbrain, cerebrum, andcerebellum) were aseptically collected from each fetus. Supernatant fromhomogenized fetal tissues was used for virus isolation attempts in cellculture. For purposes of immunohistochemistry evaluation, fetal tissueswere embedded in paraffin and tested in duplicate using 1:800 and 1:600ascites dilutions containing anti-BVDV monoclonal antibodies.

Biometric Data Analysis—To demonstrate protection following challenge, astatistically significant reduction in incidence of maternal and fetalBVDV type 2 infection had to be demonstrated in vaccinated groups (T2and T3) versus the placebo control group (T1). A Fisher's exact test wasused to compare incidence of (1) cow viremia during the first 14 daysfollowing challenge, (2) BVDV isolation from amniotic fluid, (3) BVDVisolation from fetal tissue and fetal blood following spontaneousabortion or caesarian section, and (4) BVDV-positive fetal tissueimmunohistochemistry. Serum neutralization titers were analyzed using amixed linear model with repeated measures. Least squares means from theanalysis of variance were used to calculate a geometric mean titer(GMT), which excluded SN data for cows that were not challenged. Aprobability value of P≦0.05 was used to determine statisticalsignificance.

Fetal Protection Study—The 56 test cows were randomly assigned to one ofthree test groups, an IM placebo group (T1), an IM vaccination group(T2), and a SC vaccination group (T3) as noted in Table 1. Cows wereinoculated with either vaccine or placebo on study Day 0 and Day 21. Inall cases, the Day 0 inoculation was administered on the left side ofthe neck, and the Day 21 inoculation was administered on the right sideof the neck.

On Day 1, cows were given feed top-dressed with melengestrol acetate for14 days. On Day 32, all cows received an IM prostaglandin injection(Lutalyse, Pharmacia & Upjohn, Kalamazoo, Mich.) to synchronize estrus.Cows which displayed estrus were bred by artificial insemination withcertified BVDV-negative semen. On Day 100, at approximately 65 days ofgestation, the pregnancy status of cows was determined by rectalpalpation. On Day 105, 23 cows with confirmed pregnancies were randomlyselected from each test group (7 controls, 8 IM vaccinates, and 8 SCvaccinates), relocated to a nearby isolation facility, (MidwestVeterinary Services, Oakland, Nebr.) and commingled. On Day 119, the 23test cows were challenged by intranasal inoculation of virulent BVDV.Blood samples were collected on the day of challenge and at 8 PCintervals, on Days 119, 121, 123, 125, 127, 129, 133, 140, and 147 (PCdays 0, 2, 4, 6, 8, 10, 14, 21, and 28) for purposes of BVDV isolationand serologic assay.

On Day 147 (28 days after challenge), left flank laparotomies wereperformed and amniotic fluid was extracted from each cow. On Day 297,approximately 7-14 days prior to anticipated calving, test cows weretransported to facilities at the University of Nebraska, Department ofVeterinary and Biomedical Services, for caesarian section. Immediatelyprior to surgery, a blood sample was collected from each cow for SNassay. Following caesarian delivery (on Days 300-301), a blood samplewas collected from each fetus. Fetuses were then euthanized and tissueswere aspecticaly collected for purposes of BVDV isolation.

In some cases where spontaneous abortions occurred, blood samples weretaken from the dam when abortion was detected and two weeks later. Thepaired blood samples were submitted for serologic testing (University ofNebraska Veterinary Diagnositic Center, Lincoln, Nebr.) and abortedfetuses were evaluated for BVDV isolation (Pfizer Central Research,Lincoln, Nebr.) and histopathologic evaluation of fetal tissues(Saskatoon Veterinary Biodiagnostics, Saskatoon, SK, Canada).

Results

No adverse events were observed during or immediately followingadministration of the 2 vaccine doses.

All cows were seronegative to BVDV types 1 and 2 prior to vaccination(Day 0), confirming that the test animals were immunologically naive toBVDV at the outset of the study. The GMT values for BVDV type 1 areshown in Table 2. Fifteen of 16 vaccinates seroconverted following 2vaccine doses. Cow number 61 (T3 group) had BVDV type 1 SN titers of<1:2 on Day 0, 1:19 on Day 21, and <1:2 on Day 33 and Day 119. The GMTvalues to BVDV type 2 are shown in Table 3. All vaccinates, includingcow 61, seroconverted following the second dose. (Cow 61 had BVDV type 2SN titers of <2 on Day 0, 1:19 on Day 21, 1:2,048 on Days 33, and 1:431on Day 119.) At breeding (Days 32-41), vaccinated cows (excluding cow61) had BVDV type 1 SN titers ranging from 1:64 to 1:13,777. Vaccinatedcows had BVDV type 2 titers ranging from 1:64 to 1:6,889 at abreeding.Following vaccination, the difference in GMT values for the IM (T2)versus SC (T3) groups was not statistically significant at either of 2prechallenge intervals or following challenge. All cows in the placebogroup (T1) remained seronegative for both BVDV types 1 and 2 up to thetime of challenge (Day 119), indicating that the study was notcompromised by adventitious exposure. All placebo cows respondedserologically to challenge, verifying that a viable challenge occurredin each animal. The placebo group had a PC-GMT for BVDV type 1 that wassignificantly lower than the anemnestic responses achieved by eithervaccine group (Table 2, Day 147). Placebo cows also had a lower PC-GMTto BVDV type 2 compared to either vaccine group, but the difference wasstatistically significant only versus the IM (T2) vaccinates.

Twenty-three cows in the 3 test groups were confirmed pregnant,challenged, and subjected to amniocentesis (Table 1). Between the timeof amniocentesis (Day 147) and caesarian section (Day 300-301), 7 cowsaborted, 4 from the T2 group and 3 from the T3 group. In addition, 3bred cows (1 T1 placebo cow and 2 cows from the T3 group) were found tobe not pregnant at the time of caesarian section. These 3 cows wereconfirmed pregnant by rectal palpation on Day 100, approximately 65 daysafter breeding, indicating that subsequent undetected abortion or fetalresorption occurred. Because fetal tissues from the 3 cows with failedpregnancies were not available for evaluation, these animals wereremoved from the study. On Day 259, T1 placebo cow number 67 died, andits fetus was removed for purposes of BVDV isolation. Thus, at theconclusion of the study, 12 of the 23 cows that had been challengedunderwent caesarian section. The 12 caesarian derived fetuses plus the 7aborted fetuses and the fetus from the dead cow (20 in all) wereevaluated for BVDV isolation.

Cow 38 from the T2 group aborted its fetus on Day 156 (at 123 days ofgestation, and 37 days after challenge). Paired serum samples were notevaluated, but cow 38 was negative for postchallenge BVDV isolation fromperipheral blood and amniotic fluid. The fetus was severely autolyzed.Histopathologic and bacteriologic evaluation of the fetus revealedpurulent inflammation of the chorionic and subchorionic connectivetissues. Staphylococcus hyicus was isolated from the lung, liver, andthoracic fluid. Negative BVDV isolation and immunohistochemistry resultswere obtained, indicating that the fetus was not infected as a result ofchallenge.

Three T3 cows (numbers 21, 27, and 40) aborted on Days 158 or 159 (at125-127 days of gestation, and 39 or 40 days after challenge). Theabortions were not observed, so the recovered fetuses could not beattributed to specific cows. They were designated unknown fetuses 1, 2or 3. Unknown fetus 1 was mummified, with lesions histologically typicalof neosporosis. Unknown fetus 2 was autolyzed, with multifocal purulentand necrotizing placentitis and large numbers of bacterial cocci mixedwith inflammatory exudate. Staphylococcus hyicus was isolated from lung,kidney, liver, stomach contents and placental tissue. Unknown fetus 3was macerated and autolyzed. Staphylococcus sp was isolated from thelung, liver, kidney, and stomach contents. Negative BVDV isolation andimmunohistochemistry results were obtained for all tissues from the 3unknown fetuses. All 3 dams were negative for postchallenge BVDVisolation from peripheral blood. Cows 21 and 40 were likewise negativefor BVDV isolation from amniotic fluid, but cow 27 had a BVDV-positiveamniotic fluid sample. Paired serum samples from the dams were notevaluated.

Cow 45 from the T2 group aborted its fetus on Day 160 (at 128 days ofgestation, and 41 days after challenge). Extensive fetal autolysis wasevident. Staphylococcus sp was isolated from the lung, liver, kidney,stomach contents, and placenta. Placentitis with multifocal thrombosisand suppurative vascultisis was present. Serologic assays of thoracicfluid were negative for IBR, bovine viral diarrhea (BVD), andleptospirosis. Negative BVDV isolation and immunohistochemistry resultswere obtained for all fetal tissues.

Cow 66 from the T2 group aborted its fetus on Day 195 (at 160 days ofgestation, and 76 days after challenge). Marked suppurative inflammationof the placental lamina propria extending from the fetal surface wasobserved. E. coli and Proteus vulgaris were cultured from the stomachcontents and placenta. Paired serum samples and thoracic fluid serologyresults did not support IBR, BVD, or leptospirosis as an etiology.Negative BVDV isolation and immunohistochemistry results were obtainedfor all fetal tissues.

Cow 31 from the T2 group aborted its fetus on Day 295 (at 262 days ofgestation, and 176 days after challenge). Histopathologic examinationrevealed diffuse necropurulent placentitis, necrosis of the chorionicepithelium, and intense neutrophilic inflammation. Gram-negativecoccobacilli and rods were cultured from the inflammatory foci. Pairedserology results did not support IBR, BVD, or leptospirosis as anetiology. Negative BVDV isolation and immunohistochemistry results wereobtained for all fetal tissues.

Positive BVDV isolation was obtained from PC peripheral blood samplesand amniotic fluid obtained from T1 placebo cow 67, which died prior tothe conclusion of the study. All fetal tissues from this cow were BVDVisolation and immunohistochemistry positive.

Blood samples collected from the 12 caesarian derived fetuses wereassayed for SN titers to BVDV type 1 and 2. None of the caesarianderived fetuses from the 5 placebo cows or 7 vaccinates was seropositivefor either BVDV type 1 or 2.

Postchallenge virus isolation results are shown in Table 4. All 7 T1placebo cows experienced BVDV viremia, corroborating serologic resultsindicating that a viable challenge occurred in each of the nonvaccinatedanimals. Blood samples from all 16 of the T2 and T3 vaccinates werenegative for BVDV viremia on each of 8 postchallenge samples. Thedifference in the rate of PC viremia in T2 and T3 vaccinates versuscontrols was statistically significantly (P≦0.0001).

Amniotic fluid from 2 of 16 (12.5 percent) vaccinates was BVDV positive,versus positive results for 7 of 7 (100 percent) T1 placebo cows, astatistically significant difference (P≦0.0001). Amniotic fluid samplesfrom T3 vaccinates 27 and 60 were positive. The fetus from cow 27 wasBVDV-negative by virus isolation and immunohistochemistry methods.

Fetal tissues from 1 of 14 vaccinates (7.1 percent), T3 cow 60, werepositive for BVDV isolation. This compared to BVDV isolation in 6 of 6(100 percent) fetuses from T1 placebo cows, a statistically significantdifference (P≦0.0001). BVDV isolation results were either all positiveor all negative for the fetal tissues evaluated from each fetus.

Immunohistochemistry results were BVDV positive for fetal tissues from 1of 14 (7.1 percent) T2 and T3 vaccinates (T3 cow 60). All fetal tissuesfrom 6 of 6 (100 percent) T1 placebo cows were BVDV-immunohistochemistrypositive, a significantly higher incidence (P≦0.0001) versus thevaccinates. BVDV immunohistochemistry results were either all positiveor all negative for the tissues evaluated from each fetus.

Table 5 indicates the source of virus isolation and the prechallenge SNtiters of the 2 vaccinated cows with positive BVDV isolation results.Serologic data indicates that T3 vaccinates 27 and 60 both respondedimmunologically to vaccination. Cow 27 had a BVDV type 2 SN titer atchallenge that was lower than the GMT for the T3 group, but thedifference was not significant.

Intramuscular and SC vaccination collectively provided 92.9 percentefficacy against fetal BVDV-2 infection, with negative BVDV isolationresults occurring in fetal tissues from 13 of 14 vaccinated cows (Table4). This compared to a 88.9 percent rate of protection (16 of 18vaccinates were BVDV-isolation negative) in an earlier test of the samevaccine against fetal challenge with BVDV-1 (see Example 2). In both ofthese studies, 100 percent fetal infection occurred in nonvaccinatedplacebo cows, affirming that vaccinates were exposed to a severechallenge of immunity.

Challenge virus was isolated from the amniotic fluid of 2 vaccinates,cows 27 and 60 (Table 5). As a result, both of these cows wereconsidered positive for BVDV infection, even though they were viremianegative at each of 8 PC intervals. Virus isolation, as well asimmunohistochemistry methods of high specificity and sensitivity, didnot detect BVDV in the aborted fetus from cow 27, so it was of necessityreported as BVDV-negative. Abortion of this fetus due to a BVDVinfectious process in the dam was possible.

To corroborate results, two methods were used to assess protection incows (viremia and virus isolation from amniotic fluid) and their fetuses(immunohistochemistry and virus isolation in tissue culture). The mostconservative result was used to determine rate of protection. Thus, theprotection rate in vaccinated cows was considered to be 87.5 percent (14of 16), the percentage of cows negative for virus isolation fromamniotic fluid, rather than 100 percent, the percentage ofviremia-negative cows. No published BVDV challenge-of-immunity study hasyielded 100 percent fetal protection in vaccinates against challengethat produced 100 percent fetal infection in nonvaccinated controls.Even a modified-live virus (MLV) vaccine, not commonly evaluated inpregnant cows, provided no more than 83 percent protection against fetalBVDV-1 infection in one study. (Cortese V S, Grooms, D L, Ellis J, et al(1998) Am J Vet Res. 59:1409-1413.) TABLE 1 Test groups and finalpregnancy status of cows in bovine viral diarrhea virus (BVDV) type 2fetal challenge study No. fetuses No. cows No. cows Termination ofpregnancy evaluated for vaccinated challenged (1) Material (3) Failed(4) Caesarian BVDV isolation Group Treatment (Days 0, 21) (Day 119)death^(a) (2) Abortion^(b) pregnancy^(c) section (1 + 2 + 4) T1 Placebo(IM) 18 7 1 0 1 5 6 T2 Vaccine (IM) 18 8 0 4 0 4 8 T3 Vaccine (SC) 20 80 3 2 3 6IM = intramuscular vaccination; SC = subcutaneous vaccination^(a)Maternal death occurred for T1 cow 67 (Day 259).^(b)Abortions occurred in T2 cows 38 (Day 156), 45 (Day 160), 66 (Day195), 31 (Day 295) and in T3 cows 21, 27, and 40 (Days 158 or 159).^(c)All cows with failed pregnancies were confirmed pregnant on Day 100,approximately 65 days after breeding.

TABLE 2 Bovine viral diarrhea virus (BVDV) type 1 serological responsein cows challenged with BVDV type 2 Reciprocal of BVDV type 1 serumneutralizing (SN) geometric mean titer at selected test intervals No.seropositive Treatment cows on day Vaccination Vaccination BreedingChallenge Amniocentesis Caesarian section group of breeding^(a) (Day 0)(Day 21) (Days 32-41) (Day 119) (Day 147) (Days 300-301) T1 (n = 7) 0/7<2 <2    <2 <2     65.7 833.6 (n = 6)^(d) T2 (n = 8) 8/8 <2 13.7^(b)2,383.1^(c) 480.7^(c) 1,919.2^(c) 762.7 (n = 4)^(e) T3 (n = 8)  7/8(87.5%) <2 18.2^(c) 1,116.6^(c) 570.4^(c) 1,448.3^(c) 691.7 (n = 5)^(f)T2 & T3 (n = 16) 15/16 (93.8%) <2 15.8^(c) 1,631.3^(c) 523.6^(c)1,667.2^(c) 726.3 (n = 9) ^(a)SN titer reciprocal ≧8.^(b)Statistically significant difference vs. placebo group (T1), P ≦0.0002.^(c)Statistically significant difference vs. placebo group (T1), P ≦0.0001.^(d)Cow 67 died on Day 259.^(e)Abortions occurred for cows 38 (Day 156), 45 (Day 160), 66 (Day195), and 31 (Day 295); blood was not collected from these cows on Day300-301.^(f)Abortions occurred for cows 21, 27, and 40 on Days 158 and Day 159;blood was not collected from these cows on Day 300-301.

TABLE 3 Bovine viral diarrhea virus (BVDV) type 2 serological responsein cows challenged with BVDV type 2 Reciprocal of BVDV type 2 serumneutralizing (SN) geometric mean titer at selected test intervals No.seropositive Treatment cows on day Vaccination Vaccination BreedingChallenge Aminocentesis Caesarian section group (no.) of breeding^(a)(Day 0) (Day 21) (Days 32-41) (Day 119) (Day 147) (D of breeding^(a) T1(n = 7) 0/7 <2 <2    <2 <2    402.6 2,823.8 (n = 6)^(g) T2 (n = 8) 8/8<2 7.0^(b) 1,217.7^(d) 285.2^(d) 2,598.6^(e) 922.2b (n = 4)^(h) T3 (n =8) 8/8 <2 12.8^(c)  1,837.6^(d) 261.7^(d) 1,217.5  621.3 (n = 5)^(i) T2& T3 (n = 16)   16/16 <2 9.5^(c) 1,495.9^(d) 273.2^(d) 1,778.7^(f)756.9^(b) (n = 9)^(a)SN titer reciprocal ≧8.^(b)Statistically significant difference vs. placebo group (T1), P ≦0.0064.^(c)Statistically significant difference vs. placebo group (T1), P ≦0.0005.^(d)Statistically significant difference vs. placebo group (T1), P ≦0.0001.^(e)Statistically significant difference vs. placebo group (T1), P =0.0128.^(f)Statistically significant difference vs. placebo group (T1), P =0.023.^(g)Cow 67 died on Day 259.^(h)Abortions occurred for cows 38 (Day 156), 45 (Day 160), 66 (Day195), and 31 (Day 295); blood was not collected from these cows on Day300-301.^(i)Abortions occurred for cows 21, 27, and 40 on Days 158 and Day 159;blood was not collected from these cows on Day 300-301.

TABLE 4 Summary of postchallenge cow and fetal bovine viral diarrheavirus (BVDV) isolation results Postchallenge virus isolation method andincidence Fetal tissue Treatment Viremia Anmiotic fluid Fetal tissueimmunohisto- group in cows^(a) virus isolation virus isolation^(b)chemistry^(b) T1 Placebo (IM) 7/7 (100%) 7/7 (100%) 6/6 (100%)^(c) 6/6(100%)^(c) T2 Vaccine (IM) 0/8 (0%)^(d) 0/8 (0%)^(d) 0/8 (0%)^(d) 0/8(0%)^(d) T3 Vaccine (SC) 0/8 (0%)^(d) 2/8 (25%)^(d) 1/6 (16.7%)^(c,d)1/6 (16.7%)^(c,d) T2 & T3 0/16 (0%)^(d) 2/16 (12.5%)^(d) 1/14 (7.1%)^(d)1/14 (7.1%)^(d)^(a)Virus isolation was attempted from buffy coat cell preparations fromsamples collected at 9 intervals from Day 119 (challenge) to Day 147(amniocentesis). A cow was considered viremic if any blood sample wasBVDV positive.^(b)Fetal tissues were collected following abortion or caesariansection. A fetus was considered BVDV positive if any tissues werepositive.^(c)One T1 cow, and two T3 cows were eliminated from the study becausethey were not pregnant at the time of caesarian section and abortionswere not observed.^(d)Statistically significant difference vs. placebo group (T1), P ≦0.0001

TABLE 5 Maternal serum neutralization (SN) titers and results ofchallenge in cases where bovine viral diarrhea virus (BVDV) was isolatedfrom vaccinated cows or their fetuses BVDV serotype and reciprocal SNTest Source of Termination titer at challenge group Cow no. virusisolation of pregnancy BVDV1 BVDV2 T3 27 AF Abortion 512 91 T3 60 AF,FT, IHC Caesarian section 181 152 T3 Group GMT N/A N/A 570 262AF = amniotic fluid; FT = fetal tissue; IHC = immunohistochemistry offetal tissue; GMT = geometric mean titer; NA = not applicable

EXAMPLE 2 Materials and Methods

Animals—Fifty-nine BVDV seronegative (i.e., having serum neutralization[SN] titers <1:2) cows and heifers of breeding age and soundness wereobtained from multiple sources and maintained in isolation at researchfacilities in Nebraska for the duration of the study. Each animal wasidentified with duplicate ear tags, one placed in each ear. New tagswere installed in cases where an animal lost an ear tag. Prior to thestudy, test animals were inoculated with commercial vaccines forleptospirosis, campylobacteriosis (vibriosis), and clostridialinfections. Test animals were maintained under supervision of anattending veterinarian, who clinically monitored them on a daily basis.

Test Vaccine—The test vaccine was a multivalent, modified liveinfectious bovine rhinotracheitis (IBR)-parainfluenza 3(PI3)-respiratory syncytial virus (RSV) vaccine in desiccated form,rehydrated with an inactivated, liquid BVDV vaccine(CattleMaster/PregSure 5, Pfizer Inc, New York, N.Y.). The BVDVcomponent was combined with a sterile adjuvant. Potency of the BVDVimmunizing antigen was established by calculating the geometric meantiter (GMT) for 8 replicate titrations of the bulk fluid used forvaccine preparation. Following rehydration, the IBR-PI3-BRSV-BVDVvaccine was administered in 2 mL doses by either intramuscular (IM) orsubcutaneous (SC) injection. The desiccated IBR-PI3-RSV vaccinereconstituted with sterile water was used as a placebo.

Challenge Virus—A noncytopathic BVDV type 1 field isolate (Strain816317, obtained from Dr. E. J. Dubovi, New York State College ofVeterinary Medicine, Cornell University) was used as a challenge agent.Virus identity was confirmed by SN and reverse transcriptase polymerasechain reaction (RT-PCR). The RT-PCR analysis was positive for BVDV type1 nucleotide sequences for the gp53 and p80 proteins and the 5untranslated region, and negative for the BVDV type 2 p125 sequence.Challenge virus potency was established at a GMT of 10^(4.3) TCID₅₀ permL by 2 replicate titrations made immediately before and afterchallenge. Challenge inoculum was given intranasally in a 4 mL divideddose, 2 mL per nostril.

Serologic Assays—Serum neutralization titers for BVDV types 1 and 2 weredetermined by a constant-virus, decreasing-serum assay in bovine cellculture. Serial dilutions of serum were combined with either 50-300TCID₅₀ of cytopathic BVDV type 1 strain 5960, or a similar amount ofcytopathic BVDV type 2 strain 125c.

Virus Isolation—Postchallenge isolation of BVDV in bovine cell culturewas attempted from peripheral cow blood, amniotic fluid, and fetaltissues. A BVDV-positive cell culture was determined by indirectimmunofluorescence using goat anti-BVDV polyclonal antibodies. Isolationof BVDV from fetal tissues was also attempted using immunohistochemistrymethods previously described. Haines D M, Clark E G, and Dubovi E J. VetPathol 1992; 29:27-32. Whole blood from cows was drawn from the jugularvein in 5-10 mL samples and placed in heparin-containing tubes forpreparation of buffy coat cells used for virus isolation attempts.Amniotic fluid was collected under local anesthesia by left-flanklaparotomy and aspiration of a 3-5 mL sample from the uterus. Followingcaesarian section or spontaneous abortion, the eyes, 3 brain sections,spleen, and thymus were aseptically collected from each fetus.Supernatant from homogenized fetal tissues was used for virus isolationattempts in cell culture. For purposes of immunohistochemistryevaluation, fetal tissues were embedded in paraffin and tested induplicate using 1:800 and 1:600 ascites dilutions containing anti-BVDVmonoclonal antibodies.

Biometric Data Analysis—To demonstrate protection following challenge, astatistically significant reduction in incidence of maternal and fetalinfection had to be demonstrated in vaccinated groups (T2 and T3) versusthe placebo control group (T1). A Fisher's exact test was used tocompare incidence of cow viremia and BVDV isolation from amniotic fluid,fetal tissue, and fetal tissue immunohistochemistry. Serumneutralization titers were analyzed using a mixed linear model withrepeated measures. Least squares means from the analysis of variancewere used to calculate a geometric mean titer (GMT), which excluded SNdata for cows that were not challenged. A probability value of P≦0.05was used to determine statistical significance.

Fetal Protection Study—The 59 test animals were randomly assigned to oneof three test groups, an IM placebo group (T1), an IM vaccination group(T2), and a SC vaccination group (T3) as noted in Table 6. Cows wereinoculated with either vaccine or placebo on study Day 0 and Day 21. Inall cases, the Day 0 inoculation was administered on the left side ofthe neck, and the Day 21 inoculation was administered on the right sideof the neck.

On Day 32, all cows received an IM prostaglandin injection (Lutalyse,Pharmacia & Upjohn, Kalamazoo, Mich.) to synchronize estrus. Cows whichdisplayed estrus were bred by artificial insemination with certifiedBVDV-negative semen. On Day 96, at approximately 60 days of gestation,the pregnancy status of cows was determined by rectal palpation. On Day103, 10 cows with confirmed pregnancies were randomly selected from eachtest group, and relocated to a nearby isolation facility (MidwestVeterinary Services, Oakland, Nebr.). On Day 117, these 30 cows werechallenged by intranasal inoculation of virulent BVDV. Blood sampleswere collected on the day of challenge and at 8 postchallenge intervals,on Days 119, 121, 123, 125, 127, 131, 138, and 145, for purposes of BVDVisolation.

On Day 145 (28 days after challenge), left flank laparotomies wereperformed and amniotic fluid was extracted from each cow. On Day 295,approximately 7-14 days prior to anticipated calving, test cows weretransported to facilities at the University of Nebraska, Department ofVeterinary and Biomedical Services, for caesarian section. Immediatelyprior to surgery, a blood sample was collected from each cow for SNassay. Following caesarian delivery (on Days 298-300), a blood samplewas collected from each fetus. Fetuses were then euthanized and tissueswere aspectically collected for purposes of BVDV isolation.

In cases where spontaneous abortions occurred, blood samples were takenfrom the dam when abortion was detected and two weeks later. The pairedblood samples and aborted fetuses were submitted for serologic testingof blood samples (University of Nebraska Veterinary Diagnositic Center,Lincoln, Nebr.) virus isolation from fetal tissues, (Pfizer CentralResearch, Lincoln, Nebr.) and histopathologic evaluation of fetaltissues. (Saskatoon Veterinary Biodiagnostics, Saskatoon, SK, Canada.

Results

Individual SN values for the 30 cows used in the fetal protection testwere negative for BVDV types 1 and 2 on Day 0, confirming that thesetest animals were all immunologically naive to BVDV challenge at theoutset of the study. The GMT values (Tables 7 and 8) indicate that IM(T2 group) and SC (T3 group) vaccination both elicited a serologicresponse following administration of two doses. All cows in the T2 andT3 groups seroconverted (SN titer≧1:8) to BVDV type 1 and BVDV type 2following the second vaccine dose. At breeding (Day 34-37), the BVDVtype 1 SN titers ranged from 1:27 to 1:2,900, and the BVDV type 2 titersranged from 1:609 to 1:13,777. After vaccination, GMT values for the SCgroup were marginally higher versus the IM group at each prechallengeinterval, but the differences were not statistically significant.Twenty-eight days after challenge (Day 145), the BVDV type 1 GMT values(Table 7) showed a statistically significant difference favoring the SCvaccinates (T3 group) versus the IM (T2) group.

All cows in the placebo group (T1) remained seronegative for both BVDVtypes 1 and 2 up to the time of challenge (Day 117), indicating that thestudy was not compromised by adventitious exposure. Placebo cowsresponded serologically to challenge, but their GMT responses to BVDVtypes 1 and 2 on Day 145 were significantly lower than the anemnesticresponses achieved by either vaccine group (Tables 7 and 8).

Between the time of amniocentesis (Day 145) and caesarian section (Day298-300), two cows aborted, one from the T1 group and the other from theT3 group. In addition, 4 bred cows (2 T1 placebo cows and one cow eachfrom the T2 and T3 groups) were found to be not pregnant at the time ofcaesarian section. These 4 cows were confirmed pregnant by rectalpalpation on Day 96, approximately 60 days after breeding, indicatingthat unobserved abortion or fetal resorption occurred. Because fetaltissues from the 4 cows with failed pregnancies were not available forevaluation, these animals were removed from the study. Thus, at theconclusion of the study, 24 of the 30 cows that had been challengedunderwent caesarian section, and a total of 26 fetuses resulting fromeither caesarian delivery or abortion were evaluated for BVDV isolation(Table 6).

Cow number 1317 from the T1 placebo group aborted its fetus on Day 238(after 201 days of gestation, and 121 days after challenge).Histopathologic and bacteriologic evaluation of the fetus revealedpneumonia, necrosis of the chorionic epithelium, and Corynebacterium sp.isolated from the stomach and placenta. Paired serologic samples fromthe cow did not support IBR, BVD, or leptospirosis as the abortionetiology. Positive BVDV isolation results in cell culture were obtainedfor peripheral cow blood collected at 6, 8, and 10 days after challenge;for amniotic fluid; and for fetal brain, eye, and thymus, but not thespleen. Fetal brain, eye, thymus, and spleen were immunohistochemistrypositive for BVDV. Virus isolation and serologic evidence in this caseindicates that a BVDV infected fetus was aborted by a dam thatexperienced viremia as a result of challenge.

Cow number 1331 from the T3 vaccine group aborted its fetus on Day 249(after 212 days of gestation, and 132 days after challenge).Histopathologic and bacteriologic evaluation of the fetus revealed adiffuse purulent pneumonia. Cultures of stomach contents and lung wereheavily overgrown with coliform bacteria. Paired post-abortion serologicsamples from the cow did not support IBR, BVD, or leptospirosis as theabortion etiology. Attempts at BVDV isolation in cell culture werenegative for cow peripheral blood collected at all 9 postchallengeintervals, and for amniotic fluid, and fetal brain, eye, spleen andthymus. Immunohistochemistry results for the fetal tissues werenegative. However, pooled fetal tissues were positive for BVDV isolationin cell culture. The conflicting results suggest the possibility ofcontamination of fetal tissues either by contact with pasture seededwith BVD challenge virus or by fomites at the necropsy facility, whereBVDV had been previously isolated. The absence of postchallenge viremiain the dam, its positive seroconversion status, and negative BVDVisolation results for specific fetal organs support the conclusion thatthis fetus was not BVDV infected as a result of challenge. Blood samplescollected from each caesarian derived fetus were assayed for SN titersto BVDV type 1 and 2. None of the 7 caesarian derived fetuses from T1placebo cows was seropositive for either BVDV type 1 or 2. Five of the17 caesarian derived fetuses from T2 and T3 vaccinates were seropositivefor BVDV type 1. Four fetuses had type 1 SN titers of either 1:2 or 1:4,and the fetus from T2 cow 1421 had a type 1 SN titer of 1:181 and a type2 SN titer of 1:512.

Postchallenge virus isolation results are shown in Table 9. Nine of 10T1 placebo cows experienced BVDV viremia, indicating that a viablechallenge occurred. Blood samples from 19 of the 20 T2 and T3 vaccinateswere negative for BVDV viremia on each of 8 postchallenge samples. T2vaccinate 1421 was BVDV positive on Day 123, 6 days after challenge, anddelivered a fetus that was seropositive as noted above, butvirus-isolation negative. The 5 percent incidence of postchallenge BVDVviremia in T2 and T3 vaccinates was significantly less (P≦0.0001) thanthe 90 percent rate in controls.

Fetal tissues from 2 of 18 vaccinates (11.1 percent), T2 cows 1301 and1335, were positive for BVDV isolation. This compared to BVDV isolationin 8 of 8 (100 percent) fetuses from T1 placebo cows, a statisticallysignificant difference (P≦0.0001). BVDV isolation results were eitherBVDV-positive or -negative for all fetal tissues evaluated, with oneexception, T1 cow 1317, from which 3 of 4 fetal tissue samples werepositive.

Amniotic fluid from 2 of 20 (10 percent) vaccinates was BVDV positive,versus positive results for 10 of 10 (100 percent) of T1 placebo cows, astatistically significant difference (P≦0.0001). Amniotic fluid samplesfrom T2 vaccinates 1301 and 1335 were positive.

Immunohistochemistry results were BVDV positive for fetal tissues from 2of 18 (11.1 percent) vaccinates, T2 cows 1301 and 1335. All fetaltissues evaluated from these vaccinated cows were positive. Thiscorresponded to BVDV isolation results for the same two cows when virusisolation was attempted from amniotic fluid and from fetal tissues usingcell culture methods. All fetal tissues from 8 of 8 (100 percent) T1placebo cows were BVDV positive, a significantly higher incidence(P≦0.0001) versus the vaccinates.

Prechallenge serologic status of the three vaccinated cows with positiveBVDV isolation results is shown in Table 10. Cows 1301 and 1335, whichdelivered BVDV-positive fetuses, and cow 1421, which was viremic, allresponded immunologically to vaccination.

Sixteen of 18 fetuses (88.9 percent) from vaccinated cows wererefractory to a challenge that produced 100 percent fetal infection innonvaccinated controls. The intranasal challenge not only mimicked thenatural route of infection, but at a dosage much greater than what wouldbe expected from field exposure. Challenge potency also exceeded thelevel that a prior study found would consistently achieve experimentalBVDV type 1 viremia and fetal infection. Ficken M, Jeevaraerathnam S,Wan Welch S K, et al: BVDV fetal infections with selected isolates. In:Proceedings of the International Symposium on Bovine Viral DiarrheaVirus, a Fifty-Year Review. Ithaca, N.Y., 1996; 110-112. A noncytopathicchallenge strain was used since this is the biotype associated withpersistent infection and infection of immunotolerant fetuses. Cortese VS: Bovine virus diarrhea virus and mucosal disease. In: CurrentVeterinary Therapy 4, Food Animal Practice. Philadelphia, Pa.: W BSaunders, 1999; 286-291.

Serologic data affirmed vaccine antigenicity by both IM and SC routes ofadministration. All vaccinated cows seroconverted to both BVDV types,and the marked anamnestic response to challenge (Tables 7 and 8)resulted in GMT titers that persisted until the end of the study, 6months later. The three vaccinated cows linked to positive virusisolation also seroconverted following vaccination (Table 10).Caesarian-derived calves from seropositive cows 1301 and 1335 werepositive for BVDV. Virus isolation from seropositive cows or theirfetuses suggests humoral antibody may correlate with protection but isnot its sole determinant. Cellular or mucosal mechanisms may also beinvolved. TABLE 6 Test groups and final pregnancy status of cows inbovine viral diarrhea virus (BVDV) type 1 fetal challenge study No.fetuses No. cows No. cows Termination of pregnancy evaluated forvaccinated challenged (1) Maternal (3) Failed (4) Caesarian BVDVisolation Group Treatment (Days 0, 21) (Day 119) death^(a) (2)Abortion^(b) pregnancy^(c) section (1 + 2 + 4) T1 Placebo (IM) 18 7 1 01 5 6 T2 Vaccine (IM) 18 8 0 4 0 4 8 T3 Vaccine (SC) 20 8 0 3 2 3 6IM = intramuscular vaccination; SC = subcutaneous vaccination^(a)Abortions occurred on Day 238 (T1 cow) and Day 249 (T3 cow).^(b)All cows with failed pregnancies were confirmed pregnant on Day 96,approximately 60 days after breeding.

TABLE 7 Bovine viral diarrhea virus (BVDV) type 1 serological responsein cows challenged with BVDV type 1 Reciprocal of BVDV type 1 serumneutralizing (SN) geometric mean titer at selected test intervals No.seropositive Treatment cows on day Vaccination Vaccination BreedingChallenge Amniocentesis Caesarian section group of breeding^(a) (Day 0)(Day 21) (Days 34-37) (Day 117) (Day 145) (Days 298-300) T1 (n = 10) 0/10 <2 <2    <2   <2     118.0 808.2 (n = 9)^(d) T2 (n = 10) 10/10 <25.5^(b) 414.1^(b) 177.4^(b) 10,380.1^(b) 2,233.2^(b) (n = 10) T3 (n =10) 10/10 <2 7.3^(b) 630.2^(b) 281.2^(b)  20,169.2^(b,c) 3,804.6^(b) (n= 9)^(e) T2 & T3 (n = 20) 20/20 <2 6.3^(b) 510.8^(b) 223.3^(b)14,469.2^(b) 2,914.9^(b) (n = 19)^(a)SN titer reciprocal ≧8.^(b)Statistically significant difference vs. placebo group (T1), P ≦0.003^(c)Statistically significant difference vs. IM vaccine group (T2), P =0.0446^(d)One cow aborted on Day 238.^(e)One cow aborted on Day 249.

TABLE 8 Bovine viral diarrhea virus (BVDV) type 2 serological responsein cows challenged with BVDV type 1 Reciprocal of BVDV type 2 serumneutralizing (SN) geometric mean titer at selected test intervals No.seropositive Treatment cows on day Vaccination Vaccination BreedingChallenge Amniocentesis Caesarian section group of breeding^(a) (Day 0)(Day 21) (Days 34-37) (Day 117) (Day 145) (Days 298-300) T1 (n = 10) 0/10 <1.4 <1.4    <1.4  <1.4   48.5 604.9 (n = 9)^(d) T2 (n = 10) 10/10<1.4  7.6^(b) 1,782.9^(b) 174.8^(b) 3,756.0^(b) 1,634.9^(b) (n = 10) T3(n = 10) 10/10 <1.4   17.7^(b,c) 2,749.6^(b) 309.7b 4,240.4^(b)1,879.9^(b) (n = 9)^(e) T2 & T3 (n = 20) 20/20 <1.4 11.6^(b) 2,214.1^(b)232.7^(b) 3,990.9^(b) 1,753.1^(b) (n = 19)^(a)SN titer reciprocal ≧8.^(b)Statistically significant difference vs. placebo group (T1), P ≦0.02.^(c)Statistically significant difference vs. IM vaccine group (T2), P =0.0415^(d)One cow aborted on Day 238.^(e)One cow aborted on Day 249.

TABLE 9 Summary of postchallenge cow and fetal bovine viral diarrheavirus (BVDV) isolation results Postchallenge virus isolation method andincidence Fetal tissue Treatment Viremia Amniotic fluid Fetal tissueimmunohisto- group in cows^(a) virus isolation virus isolation^(b)chemistry^(b) T1 Placebo (IM) 9/10 (90%) 10/10 (100%) 8/8 (100%)^(c) 8/8(100%)^(c) T2 Vaccine (IM) 1/10 (10%)^(d) 2/10 (20%)^(d) 2/9(22.2%)^(c,d) 2/9 (22.2%)^(c,d) T3 Vaccine (SC) 0/10 (0%)^(d) 0/10(0%)^(d) 0/9 (0%)^(d) 0/9 (0%)^(c,d) T2 & T3 1/20 (5%)^(d) 2/20(10%)^(d) 2/18 (11.1%)^(d) 2/18 (11.1%)^(d)^(a)Virus isolation was attempted from buffy coat cell preparations fromsamples collected at 9 intervals from day of challenge (Day 117) to Day145. A cow was considered viremic if any blood sample was BVDV positive.^(b)Fetal tissues were collected following abortion or caesariansection. A fetus was considered BVDV positive if any tissues werepositive.^(c)Two T1 group cows, one T2 group cow, and one T3 group cow wereeliminated from the study because they were not pregnant at the time ofcaesarian section and abortions were not observed.^(d)Statistically significant difference vs. placebo group (T1), P ≦0.008.

TABLE 10 Cow pre-challenge serum neutralization (SN) titers in caseswhere bovine viral diarrhea virus (BVDV) was isolated from vaccinatedcows or their fetuses BVDV serotype and reciprocal Test Source of SNtiter at challenge group Cow no. virus isolation BVDV1 BVDV2 T2 1301 FT,AF, IHC 128 181 T2 1335 FT, AF, IHC 109  91 T2 1421 CV   64^(a)   27^(a)T2 Group GMT N/A  177.4  174.8FT = fetal tissue; AF = amniotic fluid; IHC = immunohistochemistry offetal tissue; CV = cow viremia; NA = not applicable; GMT = geometricmean titer

EXAMPLE 3

Two groups of 16 cattle were vaccinated twice subcutaneously at aninterval of 3 weeks with 2 mL of L. hardjo/L. pomona combinationvaccines prepared from two adjuvant formulations: 1) 2.5% Amphigen withQuil A/cholesterol each at 250 mcg/mL, and 2) Amphigen/AI-gel. Thevaccines consisted of killed leptospires from which the culture fluidshad been removed, so free endotoxin was low. Body temperatures,injection-site reactions, and general health observations were recordedfollowing both injections. No systemic affects were seen, and localreactions were minimal and judged to be clinically acceptable. Sixteenadditional cattle were injected with saline as controls. Four weeksafter vaccination, cattle were challenged by ocular and vaginalinstillation of 5×10⁶ leptospires on 3 consecutive days. Half of eachtreatment group was challenged with serovar hardjo and half with pomona.Two pomona controls were eliminated from the study for unrelatedreasons, leaving 6 animals in that group. Urine collected weekly, andkidney samples collected at necropsy, 8 weeks after challenge, wereevaluated by culture, PCR, and fluorescent antibody microscopy (FA).

Following L. hardjo challenge, viable organisms were detected in urineand/or kidney cultures from 100% (8/8) of the unvaccinated controls,whereas positive cultures were never obtained from any vaccinated animal(0/16). After challenge with L. pomona, 67% (4/6) of the unvaccinatedcontrols became infected on the basis of urine/kidney culture, but noneof the vaccinates was ever kidney or urine culture positive (0/16).

Since leptospirosis is transmitted via contaminated urine, the abilityto prevent or reduce urinary shedding is a useful measure of vaccineefficacy. Both vaccine formulations reduced urinary shedding ofleptospires by statistically significant amounts compared to controls.The data show a substantial benefit from vaccination with the bivalentL. hardjo/L. pomona vaccines formulated with either adjuvant;demonstrating the protection of cattle from infection with leptospiresby vaccinating with formalin-killed combination bacterins.

EXAMPLE 4 Materials and Methods

Animals—Thirty-six BVDV and Leptospira seronegative (i.e., having BVDVserum neutralization [SN] titers<1:2 and Leptospira serovars hardjo andpomona [MAT] titers<1:20)) approximately 7-month old calves wereobtained from multiple sources and maintained in isolation at researchfacilities in Nebraska for the duration of the study. Each animal wasidentified with duplicate ear tags, one placed in each ear. New tagswere installed in cases where an animal lost an ear tag. Prior to thestudy, test animals were vaccinated against clostridial diseases andbovine respiratory disease agents (excluding BVD virus). Test animalswere maintained under supervision of an attending veterinarian, whoclinically monitored them on a daily basis.

Test Vaccines—The experimental test vaccines were liquid vaccinescontaining either formalin inactivated L. hardjo-bovis or L. pomona, orboth, and inactivated BVD type 1 and type 2 viruses. The BVDV componentswere combined with a sterile adjuvant. Potency of the BVDV immunizingantigens was established by calculating the geometric mean titer (GMT)for 8 replicate titrations of the bulk fluid used for vaccinepreparation. Potency of the Leptospira immunizing antigens wasestablished in accordance with a hamster lethality model procedure. Theadjuvants in the experimental test vaccines were comprised of either2.5% Amphigen (v/v) with Quil A/cholesterol each at 100 mcg/ml, with orwithout 2% (v/v) aluminum hydroxide; 2.5% Amphigen (v/v) with QuilA/Dimethyl dioctadecylammonium bromide (DDA) each at 100 mcg/ml, with orwithout 2% (v/v) aluminum hydroxide. The experimental test vaccines wereadministered in 5 mL dose by subcutaneous (SC) injection. A monovalentL. hardjo-bovis bacterin was used as a positive control permanufacturer's instructions. A placebo vaccine containing physiologicalsaline was used as a negative control.

Challenge Bacteria—Leptospira borgpetersenii serovar hardjo typehardjo-bovis strain 203 (National Animal Disease Center, Ames, Iowa),was used as the challenge agent. L. hardjo-bovis challenge material wasprepared as first passage organisms which had been isolated from theurine of cattle experimentally infected with L. hardjo-bovis. Thechallenge material was administered once daily for three consecutivedays. Each challenge day, a total of two mL of challenge material,containing approximately 2.5×10⁶ L. hardjo-bovis organisms/mL, wasadministered across three separate anatomical sites. The route ofchallenge was instillation into the conjunctival sac of each eye (½ mLeach) and into the vagina (1 mL).

Serologic Assays—Serum neutralization titers for BVDV types 1 and 2 weredetermined by a constant-virus, decreasing-serum assay in bovine cellculture. Serial dilutions of serum were combined with either 50-300TCID₅₀ of cytopathic BVDV type 1 strain 5960, or a similar amount ofcytopathic BVDV type 2 strain 125c. Serum microscopic agglutinationtiters (MAT) for L. hardjo-bovis and L. pomona were conducted using astandard test at a qualified veterinary diagnostic center (CornellUniversity College of Veterinary Medicine Diagnostic Laboratory).

Leptospira Isolation—Urine samples and kidney tissue homogenates (pooledleft and right kidney) were examined for the presence of Leptospira.Urine and kidney cultures were examined for Leptospira once weekly forup to 8 weeks using standard procedures. Leptospira fluorescent antibody(FA) techniques were conducted using a standard test at a qualifiedveterinary diagnostic center (Cornell University College of VeterinaryMedicine Diagnostic Laboratory).

Biometric Data Analysis—To demonstrate protection following challenge, astatistically significant reduction in incidence of Leptospira infectionhad to be demonstrated in vaccinated groups (Table 11) (T02, T03, T04and T05) versus the placebo control group (T1). Data for kidneycolonization and urinary shedding were summarized by treatment andtimepoint. Comparisons between treatments were made as to the percent ofanimals with Leptospira detected in the kidney. Comparisons betweentreatments were made as to the percent of animals with Leptospiradetected in the urine. A Fisher's Exact test was used for the analysesabove. Duration of Leptospira shedding in the urine was also comparedusing a general linear mixed model. A probability value of P≦0.05 wasused to determine statistical significance.

Leptospira Protection Study—The 36 test animals were randomly assignedto one of six test groups as indicated in Table 11. On Day 0 and Day 21,each animal assigned to T01-T05 received one 5 mL SC dose of theappropriate experimental test or placebo vaccine. On Day 0 and Day 28,each animal assigned to T06 received one 2 mL SC dose of the positivecontrol vaccine. On Days 57-59, all animals were challenged with L.hardjo-bovis strain 203 as outlined above.

Blood samples were collected from each animal on Days 0, 21, 35, 56, 84,and 111 for determination of BVDV type 1 and type 2 titers.

Urine samples (approximately 45 mL) were collected from each animal onDays −1, 56, 70, 77, 84, 91, 98 and 105 for leptospire isolation asdescribed above.

Animals were euthanized on Days 112 and 113 and kidneys evaluated forthe presence of leptospires as described above.

Results

The GMT values (Table 12) indicate that all the animals receivingBVDV-Leptopsira combination vaccines (T02, T03, T04 and T05) elicited aserologic response following administration of two vaccine doses. Allthe animals in groups T02, T03, T04 and T05 seroconverted (SN titer≧1:8)to BVDV type 1 following the second vaccine dose. All the animals ingroups T02, T04 and T05 seroconverted (SN titer≧1:8) to BVDV type 2following the second vaccine dose. All cows in the placebo group (T01)or in the group that received monovalent L. hardjo-bovis vaccineremained seronegative from both BVDV types 1 and 2, indicating that thestudy was not compromised by adventitious exposure. Collectively, theBVDV serology data shows for the first time that combination vaccinescomprising inactivated BVDV types 1 and 2 and inactivated L. hardjobovisand L. pomona formulated in four different adjuvants can induce aprotective response against BVDV disease in cattle, since a cow BVDV SNtiter of ≧1:8 is known in the art to be indicative of protection againstBVDV disease.

The Leptospira urine and kidney results (Table 13) indicate that allanimals that received BVDV-Leptospira combination vaccines (T02, T03,T04 and T05) were urine culture (CX) negative (Table 13, column 2) atall eight timepoints tested and kidney culture negative (Table 13,column 8) at necropsy (Day 112 or 113). Cows that received Leptospiramonovalent vaccine (T06, positive control) were similarly protectedagainst Leptospira infection. In contrast, cows that received placebovaccine (T01, negative control) were infected based on urine (Table 13,column 2) and kidney culture (Table 13, column 8), indicating thevaccine challenge study was valid. Collectively, the L. hardjobovisisolation data shows for the first time that combination vaccinescomprising inactivated BVDV types 1 and 2 and inactivated L. hardjobovisand L. Pomona formulated in four different adjuvants can induce aprotective response against Leptospira disease in cattle. TABLE 11 Testgroups of calves in BVDV-Leptospira combination vaccine study DosingNumber of Dose Route of Number of Treatment Interval Vaccine AnimalsVolume Administration Doses/Animal T01 3 weeks Saline placebo 6 5 mL SC2 T02 3 weeks L. hardjobovis- 6 5 mL SC 2 L. Pomona-BVDV-1- BVDV-2 inQAC T03 3 weeks L. hardjobovis- 6 5 mL SC 2 L. Pomona-BVDV-1- BVDV-2 inQAC/AIOH T04 3 weeks L. hardjobovis- 6 5 mL SC 2 L. Pomona-BVDV-1-BVDV-2 in DDA T05 3 weeks L. hardjobovis- 6 5 mL SC 2 L. Pomona-BVDV-1-BVDV-2 in DDA/AIOH T06 4 weeks L. hardjobovis 6 2 mL SC 2 monovalent

TABLE 12 BVDV Types 1 and 2 Serum Neutralization Reciprocal GeometricMean Titers and Ranges (#-#) on Day 35 Serum Virus NeutralizationReciprocal Geometric Mean Titer and Range (#-#) on Day 35 Treatment BVDVirus Type 1 BVD Virus Type 2 T01 <2 (<2-<2) <2 (<2-<2) T02 1084.9(609-2435) 20.8 (16-54) T03 148.0 (<2-1218) 5.8 (<2-19) T04 1877.9(1024-2896) 34.0 (19-91) T05 1084.6 (152-3444) 36.1 (10-91) T06 <2(<2-<2) <2 (<2-<2)

TABLE 13 Efficacy Results of BVDV-Leptospira Combination VaccinesAgainst Leptospira hardjo-bovis Challenge Percent of Calves Ever LeastSquares Mean Percent Percent of Calves Ever Positive Positive forLeptospira in Urine Days of Leptospira Positive Urine for Leptospira inKidneys Treatment CX FA PCR CX FA PCR CX FA PCR T01 n = 6 100^(a)  83.3^(a)  83.3^(a)  50.9^(a)  29.1^(a)  37.1^(a)  83.3^(a) 0^(a) 16.7^(a) T02 n = 6 0^(b) 0^(b) 0^(b) 0^(b) 0^(b) 0^(b) 0^(b) 0^(a)0^(a) T03 n = 6 0^(b) 0^(b) 0^(b) 0^(b) 0^(b) 0^(b) 0^(b)  50.0^(a)0^(a) T04 n = 6 0^(b) 0^(b) 0^(b) 0^(b) 0^(b) 0^(b) 0^(b) 0^(a) 0^(a)T05 n = 6 0^(b)   16.7^(ab)   16.7^(ab) 0^(b)  0.3^(b)  0.4^(b) 0^(b) 16.7^(a) 0^(a) T06 n = 6 0^(b) 0^(b)   33.3^(ab) 0^(b) 0^(b)  1.6^(b)0^(b) 0^(a) 0^(a)Values within a column not sharing a common superscript weresignificantly (P < 0.05) different

EXAMPLE 5 Materials and Methods

Animals—Twenty BVDV seronegative (i.e., having serum neutralization [SN]titers<1:2) cows were obtained and maintained in research isolationfacilities for the duration of the study. Each animal was identifiedwith duplicate ear tags, one placed in each ear. New tags were installedin cases where an animal lost an ear tag. Test animals were maintainedunder supervision of an attending veterinarian, who clinically monitoredthem on a daily basis.

Test Vaccine—The test vaccine was a multivalent, modified liveinfectious bovine rhinotracheitis (IBR)-parainfluenza 3(PI3)-respiratory syncytial virus (RSV) vaccine in desiccated form,rehydrated with an inactivated, liquid 8-way BVDV-Leptospiraspp-Campylobacter fetus containing vaccine in a QuilA/cholesterol/Amphigen adjuvant. (Pfizer Inc, New York, N.Y.) The liquidcomposition consisted of inactivated BVDV-1 and -2 viruses, fiveinactivated Leptospira species (L. canicola, L. grippotyphosa, L.borgpetersenii hardjo-prajitno, L. icterohaemorrhagiae and L.interrogans pomona) and inactivated C. fetus bacterin combined with aQuil A/cholesterol/Amphigen adjuvant sterile adjuvant. Control vaccineconsisted of the five inactivated Leptospira spp. described above andinactivated C. fetus bacterin combined with a sterile mineral oil(Drakeol) adjuvant. Test vaccine was given by subcutaneous injection andcontrol vaccine was given by intramuscular injections. Vaccines wereadministered on Day 0 on the right side of the neck and on Day 21 on theleft side of the neck.

Challenge Virus—A cytopathic BVDV type 2 field isolate (Strain 24515)was used as a challenge agent. Challenge virus potency was establishedat a GMT of 10^(5.4) TCID₅₀/5 mL by 2 replicate titrations madeimmediately before and after challenge. Challenge inoculum was givenintranasally on Day 42 in a 5 mL divided dose, approximately 2.5 mLs pernostril.

Serologic Assays—Serum neutralization titers for BVDV types 1 and 2,BHV-1, PI3 and BRSV were determined by a constant-virus,decreasing-serum assay in bovine cell culture using standard procedures.C. fetus antibody titers were determined by a standard agglutinationassay.

Virus Isolation—Postchallenge (PC) isolation of BVDV in bovine cellculture was conducted from peripheral white blood cells (buffy coats) onDays 42 and 45-52. A BVDV-positive cell culture was determined byindirect immunofluorescence using goat anti-BVDV polyclonal antibodies.Whole blood from cows was drawn from the jugular vein in 5-10 mL samplesand placed in heparin-containing tubes for preparation of buffy coatcells used for virus isolation attempts.

Clinical Disease Scoring—Each animal was scored on Days 40-56post-challenge. A normal animal with no clinical signs received a scoreof zero. An animal with nonspecific clinical signs (eg nasal discharge,abnormal respiration, and lethargy) not specific for acute BVD virusinfection received a score of one. A score of two was assigned to anyanimal with acute BVD clinical disease in which clinical signs as awhole were moderate and specific for acute BVD virus infection. Clinicalsigns include nasal discharge, abnormal respiration, lethargy,gauntness, ocular discharge, hypersalivation, diarrhea, dehydration,lameness and/or reluctance to move. An animal with clinical signs thatas a whole were severe in degree was assigned a score of three.

Biometric Data Analysis—For serum virus neutralization, titers weretransformed to log base 2 and analyzed by a mixed linear model withrepeated measures. Backtransformation was done to calculate geometricmean titer (GMT). Percent number of days with positive virus isolationwas analyzed using a mixed linear model. Pairwise comparison of testversus control vaccine groups were made. A probability value of P≦0.05was used to determine statistical significance.

Results

No adverse events were observed during or immediately followingadministration of the 2 vaccine doses.

All cows were seronegative to BVDV types 1 and 2 and BHV-1 prior tovaccination (Day 0), confirming that the test animals wereimmunologically naïve to BVDV and BHV-1 at the outset of the study. TheGMT values for all five viral fractions on Day 0 and Day 35 (14 dayspost-second vaccination) are shown in Table 14. TABLE 14 Serum viralneutralization titers to BVDV type 1, BVDV type 2, BHV-1, PI3 and BRSVand agglutination antibody titers to C. fetus prior to (Day 0) andfollowing vaccine administration. Vaccine BVDV-1 BVDV-2 BHV-1 PI3 BRSVC. fetus Vaccine Components 0 35 0 35 0 35 0 35 0 35 0 35 Control 5Leptospira 1 1^(a) 1  1^(a) 1 1^(a) 140 175 8 14 57 260 spp., C. fetusTest BHV-1, PI3 1 8^(b) 1 18^(b) 1 145^(b)  155 453 16 57 26 422 BRSV,BVDV-1 BVDV-2 5 Leptospira spp., C. fetus^(a,b)Significant differences between vaccine treatments within a columnare indicated by different subscripts

Results show that the 11-way test vaccine composition was immunogenic incattle since differences in antibody titers to all 5 viruses wereobserved between pre-vaccination (Day 0) and post-vaccination (Day 35)timepoints. In addition, post-vaccination (Day 35) titers to C. fetuswere similar between the 5-way control and 11-way test vaccine,demonstrating that the presence of the modified-live and killed viralfractions in the 11-way vaccine did not interfere with ability of thehost to mount an immune response against the C. fetus bacterialfraction.

EXAMPLE 6 Materials and Methods

Animals—Thirty male and female calves were selected for the study from asingle herd. The ages of these calves were estimated to be 6 to 8 monthsbased on their body weights on Day 0. An additional ten calves from thesame herd [six were not] were enrolled on Day 18; however, they had notbeen weighed. Prior to being enrolled in the study (Day 0 for T02, T03,T04, and Day 18 for T01) all animals were seronegative (SVN<1:2) forantibodies to BVD virus Type 1 and Type 2. Each animal was identifiedwith duplicate ear tags, one placed in each ear. New tags were installedin cases where an animal lost an ear tag. Test animals were maintainedunder supervision of an attending veterinarian, who clinically monitoredthem on a daily basis.

Test Vaccine—The test vaccines were prepared by reconstituting thelyophilized modified live virus vaccine CattleMaster™, containingmodified live infectious bovine rhinotracheitis (IBR)-parainfluenza 3(PI3)-respiratory syncytial virus (RSV) with one of the killed BVDliquid diluents (containing BVDV types 1 and 2) prepared in amicrofluidized saponin-based (Quil A or GPI-0100) oil-in-water emulsion.The method for preparation of a microfluidized saponin-basedoil-in-water emulsion is described in Application Ser. No. 60/460,301,filed Apr. 4, 2003, incorporated herein by reference. (Treatment groupsare shown in Table 15). TABLE 15 Treatment Groups used in Saponin-basedOil-in-Water Emulsion Vaccine Efficacy Study Saponin # of Group # ofcalves Vaccine Fractions Adjuvant Doses T01 10 0.9% saline none 2 T02 10Modified-live BHV-1 Quil A 2 Modified-live PI3 Modified-live BRSV 0.5mg/dose Killed BVDV 1 Killed BVDV 2 T03 5 Modified-live BHV-1 GPI-0100 2Modified-live PI3 Modified-live BRSV 0.5 mg/dose Killed BVDV 1 KilledBVDV 2 T03 15 Modified-live BHV-1 GPI-0100 2 Modified-live PI3Modified-live BRSV 1.0 mg/dose Killed BVDV 1 Killed BVDV 2

Vaccines were administered as a single 2 mL dose subcutaneously (SC) onthe right side of the neck for the first administration (Day 0 and/orDay 2) and on the left side of the neck for the second administration(Day 21). Injections were administered in the lateral neck approximatelymidway between the scapula and the poll.

Challenge Virus—The challenge virus was non-cytopathic Bovine ViralDiarrhea virus (BVDV) Type 2, strain 24515. On Day 42 a 5 mL dose of thechallenge virus preparation (approximately 2.5 mL per nostril) wasadministered intranasally (needle-less syringe administration) toanimals in treatments T01, T02, T03 and T04. The challenge material wastitrated for virus content (two replicates per assay) prior to andfollowing challenge administration. The mean titers pre-challenge andpost-challenge were 5.5 log₁₀ and 5.3 log₁₀ per 5 mL dose, respectively.

Serologic Assays—Blood samples (two 13 mL SST tubes) for BVD serologywere collected o Days 0, 21, 35, 43, and 57. Serum neutralization titersfor BVDV types 1 and 2, BHV-1, were determined by a constant-virus,decreasing-serum assay in bovine cell culture using standard procedures.

Total White Blood Cell (WBC) Counts: Blood samples (One 4 mL EDTA tube)for total WBC determination were collected from T01-T04 animals on Days41, 42 and 43 (prior to challenge and following challenge) and on Days44 through 57. Blood samples were processed and transferred toPhysicians Laboratory Services, Inc. for analysis. The results weretransferred electronically into a Clinical Data Management System

Virus Isolation—Blood samples (one 8 mL CPT tube) for BVD virusisolation were collected from T01-T04 on Day 43 (prior to challenge) andon Days 44-57. A BVDV-positive cell culture was determined by indirectimmunofluorescence using goat anti-BVDV polyclonal antibodies

Clinical Disease Scoring—Clinical disease scores of 0, 1, 2, or 3 basedon clinical signs attributable to BVD 2 infection (see above, example4), were made for each animal T01-T04 on Days 41 through 43 (prior tochallenge) and Days 44 through 57.

Biometric Data Analysis—For serum virus neutralization, titers weretransformed to log base 2 and analyzed by a mixed linear model withrepeated measures. Backtransformation was done to calculate geometricmean titer (GMT). Percent number of days with positive virus isolationwas analyzed using a mixed linear model. Pairwise comparison of testversus control vaccine groups were made. A probability value of P≦0.05was used to determine statistical significance.

Results

No untoward systemic reactions were observed in any of the animalsduring the vaccination phase of the study (Days 0 through 42).

The geometric mean reciprocal SVN titers for antibodies to the BVD virusType 1 and Type 2 are summarized in Tables 16 and 17. TABLE 16 GeometricMean SVN Titers for Antibodies to BVD Virus Type 1 BVD-1 Geometric MeanReciprocal SVN Titers on Study Day Treatment N 0 21 35 43¹ 57 T01 saline10 NS¹ <2^(b  )   <2^(b)  <2^(b)   43.6^(b) T02 Quil A, 0.5 mg 10 <212.5^(a  ) 2393.6^(a) 2797.9^(a) 31651.6^(a) T03 GPI, 0.5 mg 5 <222.1^(a,c) 8480.8^(c) 7912.9^(c) 92682.0^(a) T04 GPI, 1.0 mg 15 <224.6^(a,c) 6968.7^(c) 6136.9^(c) 61857.7^(a)

TABLE 17 Geometric Mean SVN Titers for Antibodies to BVD Virus Type 2BVD-2 Geometric Mean Reciprocal SVN Titers on Study Day Treatment N 0 2135 43² 57 T01 saline 10 NS¹ <2^(b) <2^(b)  <2^(b)   494.6^(b) T02 QuilA, 0.5 mg 10 <2   4.0^(a) 469.4^(a) 587.1^(a) 75281.2^(a) T03 GPI, 0.5mg 5 <2 <2^(b) 100.3^(c)  87.4^(c) 18820.1^(c) T04 GPI, 1.0 mg 15 <2<2^(b) 125.5^(b) 100.1^(c) 18604.0^(c)NS = Animals not on studyValues for each day either with no superscripts or the same superscriptwere not statistically significant, P≧0.05

As shown in Tables 16 and 17, all three saponin containing oil-in wateremulsion adjuvants induced statistically significant antibody titers toBVDV type 1 and type 2 viruses on Days 21, 35, 43, and 57. Collectively,these data demonstrate that vaccine compositions comprisingmodified-live BHV-1, PI3, BRSV and at least one additional antigen andan adjuvant that comprises a saponin containing oil-in-water emulsion isimmunogenic in cattle. In addition, these data demonstrate such vaccinescomprising saponin containing oil-in-water microfluidized emulsion asthe adjuvant are immunogenic in cattle.

1.-83. (canceled)
 84. A vaccine composition comprising a Bovine ViralDiarrhea Virus (BVDV) and a veterinary-acceptable carrier.
 85. Thevaccine composition according to claim 84, wherein said BVDV is amodified live vaccine.
 86. The vaccine composition according to claim84, wherein said BVDV is inactivated.
 87. The vaccine compositionaccording to claim 85, wherein said BVDV is Bovine Viral Diarrhea VirusType-1 (BVDV-1) or Bovine Viral Diarrhea Virus Type-2 (BVDV-2).
 88. Thevaccine composition according to claim 86, wherein said BVDV is BovineViral Diarrhea Virus Type-1 (BVDV-1) or Bovine Viral Diarrhea VirusType-2 (BVDV-2).
 89. The vaccine composition according to claim 87,wherein said BVDV-1 and BVDV-2 are cytopathic.
 90. The vaccinecomposition according to claim 87, wherein said BVDV-1 and BVDV-2 arenoncytopathic.
 91. The vaccine composition according to claim 84,further comprising an adjuvant.
 92. An immunogenic compositioncomprising a Bovine Viral Diarrhea Virus (BVDV) and aveterinary-acceptable carrier.
 93. The immunogenic composition accordingto claim 92, wherein said BVDV is a modified live vaccine.
 94. Theimmunogenic composition according to claim 92, wherein said BVDV isinactivated.
 95. The immunogenic composition according to claim 93,wherein said BVDV is Bovine Viral Diarrhea Virus Type-1 (BVDV-1) orBovine Viral Diarrhea Virus Type-2 (BVDV-2).
 96. The immunogeniccomposition according to claim 94, wherein said BVDV is Bovine ViralDiarrhea Virus Type-1 (BVDV-1) or Bovine Viral Diarrhea Virus Type-2(BVDV-2).
 97. The immunogenic composition according to claim 95, whereinsaid BVDV-1 and BVDV-2 are cytopathic.
 98. The immunogenic compositionaccording to claim 95, wherein said BVDV-1 and BVDV-2 are noncytopathic.99. The immunogenic composition according to claim 92, furthercomprising an adjuvant.
 100. A method of preventing fetal infection inan animal subject, comprising administering to said animal an effectiveamount of the vaccine composition of claim
 84. 101. The method accordingto claim 100, wherein said fetal infection is persistent fetalinfection.
 102. The method of claim 100, wherein said animal is a cow, acalf, a heifer, a steer or a bull.
 103. The method of claim 102, whereinsaid animal is a lactating cow.
 104. The method of claim 102, whereinsaid animal is a pregnant cow.
 105. The method of claim 102, whereinsaid animal is a prebreeding cow or heifer.
 106. The method of claim100, wherein the amount of said vaccine administered is from about 0.5to about 5.0 ml per dose.
 107. The method of claim 106, wherein theamount of said vaccine administered is about 5 ml per dose.
 108. Themethod of claim 106, wherein the amount of said vaccine administered isabout 2 ml per dose.